Antibody derivatives

ABSTRACT

The invention relates inter alia to a bivalent, bispecific construct comprising an anti-IL-6 antibody, or derivative thereof, and an anti-IL-23 antibody, or derivative thereof and its use in therapy. The invention also relates to useful anti-IL-6 antibodies and anti-IL-23 antibodies.

RELATED APPLICATIONS

The present application is a Continuation of co-pending PCT ApplicationNo. PCT/EP2011/065697 filed Sep. 8, 2011, which in turn, claims priorityfrom U.S. Provisional Application Ser. No. 61/381,789, filed Sep. 10,2010. Applicants claim the benefits of 35 U.S.C. §120 as to the PCTapplication and priority under 35 U.S.C. §119 as to the said U.S.Provisional application, and the entire disclosures of both applicationsare incorporated herein by reference in their entireties.

This disclosure relates to novel antibody derivatives, methods forpreparing them, compositions containing them, and their use in therapy.

INTRODUCTION Bispecific Antibodies

Monospecific antibodies, such as the naturally-occurring IgG, have twoidentical antigen binding paratopes, as they are made of two identicalheavy chains and two identical light chains. Bispecific antibodies areengineered immunoglobulin derivatives that have two different bindingparatopes, usually directed at different antigens or epitopes.

Recombinant bispecific antibodies (“bispecifics”) have been developedfor a variety of different applications with potential use in cancertherapy, inflammatory conditions, and thrombolytic therapy, to name afew. In cancer therapy, these applications include the retargeting ofeffector molecules (prodrug-converting enzymes, radio-isotopes,complement components), effector cells (CTLs, NK cells), and thedelivery of prodrugs or chemotherapeutic agents. In the context ofinflammation, bispecifics have been developed to inhibit two or morecytokines. Recent work explored their use as intracellular bispecificantibodies (intrabodies) (Kontermann and Müller, 1999). In one study anintracellularly expressed diabody was used to inhibit functionalexpression of two cell surface receptors (Jendreyko et al., 2003).

Bispecifics must have strong and selective binding to a disease-relatedantigen, and are designed to be non-immunogenic by a variety oftechniques, such as antibody humanization, the use of transgenichumanized mice, or de-immunization. The widespread development ofbispecific antibodies has been hampered by the difficulty of producingmaterials of sufficient quality and in sufficient quantity for in vivopreclinical and clinical studies. Traditionally, efficient bispecificantibody production has required both a novel structural format thatenables the formation of stable homogenous bispecific proteins, and anefficient expression system that leads to high-level production. Avariety of approaches have been used to generate bispecific antibodiesin prokaryotic and/or eukaryotic systems, primarily involving geneticfusion of the antigen binding domains. Some limited efforts usingchemical conjugation have also been tested.

Stability of the recombinant bispecific antibodies under storageconditions as well as the stability and half life of these molecules invivo are critical parameters with strong impact for clinicalapplication. The bispecific has to be sufficiently stable to allow themolecules to induce a therapeutic benefit before being degraded. Severalstudies showed that tandem scFv molecules, as well as diabodies, wereinactivated under physiological conditions, with varying half-livesdepending on the antibody construct tested. One approach to improve thestability of antibody molecules is the generation ofdisulfide-stabilized molecules introducing cysteine bridges between theVH-VL interfaces to inhibit dissociation of the VH and VL domains.However, a marked reduction in production yield has been reported forthese disulfide-stabilized bispecific diabodies in E-coli. As such thereis a need for further bispecific constructs having improved stability,half lives and yields that are suitable for therapeutic applications.

Interleukins and their Role in TH Mediated Responses

CD4⁺ T-helper (T_(H)) lymphocytes represent a heterogeneous populationof cells that play an essential role in adaptive immunity. These cellsinclude effector cells, which are devoted to protection againstpathogens, and regulatory T cells (Tregs), which protect againsteffector responses to autoantigens. The term T_(H) derived from theobservation that these cells are critical for helping B cells to produceantibodies. On the other hand, CD4⁺ T cells were also found to beresponsible for helping CD8⁺ T cells differentiate into killer effectorcells of the so-called cell-mediated immunity. CD4⁺ T cells maythemselves be immune effector cells in immune reactions such asdelayed-type hypersensitivity, in which these cells induce inflammatoryreactions mainly characterized by the activation of macrophages.

Two decades ago, two T helper cell subsets were described. T_(H)1 cellsproduce IFNγ and their primary role is the protection againstintracellular microbes, while T_(H)2 cells produce IL-4, IL-5, and IL-13and are historically associated with atopy and asthma. T_(H)1 and T_(H)2cell development are under the control of certain transcription factorsincluding T box expressed in T cells (Tbet) and signal transducer andactivator of transcription (STAT) 4 for T_(H)1 cells and GATA-bindingprotein (GATA)-3 and STATE for T_(H)2 cells.

T_(H)1 differentiation is mainly driven by IL-12 and IFNγ, while IL-4(in the absence of IL-12) drives T_(H)2 differentiation. In CD4⁺ Tcells, IL-12 signaling, along with antigen presentation, is believed toshift cell differentiation toward the T helper (T_(H)) 1 phenotype, andis associated with robust production of the proinflammatory cytokine,interferon gamma (IFN-γ).

A recently described third subset of T helper cells, T_(H)17 cells, isabundant at mucosal interfaces, where they contain infection withpathogenic bacteria and fungi. These cells produce IL-17A (also referredto as IL-17), IL-17F, and IL-22, cytokines involved in neutrophilia,tissue remodeling and repair, and production of antimicrobial proteins.The differentiation of T_(H)17 is somewhat controversial: the currentconsensus is that IL-1 and IL-6 induce early T_(H)17 differentiationtogether with TGF-β. It has been reported that IL-21, similar to IL-2,acts as a growth factor for T_(H)17. The combination of IL-6 and TGF-βinduces the orphan nuclear receptors, retinoid related orphan receptor(ROR) γt and RORα, which are the key transcription factors indetermining the differentiation of the T_(H)17 lineage as well as theIL-23R. STAT3 regulates IL-6-induced expression of RORγt and RORα andIL-17 production. In contrast to STAT3 activation, STAT1 activationinhibits the development of T_(H)17 cells. Although IL-6 activates bothSTAT3 and STAT1, it has been demonstrated that STAT3 activation ismaintained while STAT1 activation is suppressed in T_(H)17 cells. IL-23has been implicated in the maintenance and activation of human T_(H)17cells.

IL-22 was originally described in mice and humans as a cytokinecharacteristic of fully differentiated T_(H)17 cells. Recently, however,a distinct subset of human skin-homing memory T cells has been shown toproduce IL-22, but neither IL-17 nor IFNγ. Differentiation of IL-22producing T cells, now named T_(H)22 cells, could be promoted bystimulation of naive T cells in the presence of IL-6 and TNF or by thepresence of plasmacytoid dendritic cells, and appears to be independentof RORC. The human T_(H)22 cell population coexpresses the chemokinereceptor CCR6 and the skin-homing receptors CCR4 and CCR10, which led tohypotheses that these cells may be important in skin homeostasis andpathology.

T_(H)1 cells were long considered to be the major effectors in multipleautoimmune diseases, while T_(H)2 cells have been known to be involvedin atopy and asthma. More recently, T_(H)17 cells have been implicatedas culprits in a plethora of autoimmune and other inflammatory diseasesin mice and humans. Many of the disease states previously associatedwith T_(H)1 cells, e.g., experimental autoimmune encephalomyelitis (EAE,a model for multiple sclerosis), collagen-induced arthritis, and someforms of colitis, were shown to be caused by IL-23-dependent T_(H)17cells or other IL-17-producing lymphoid cell types. An imbalance betweenT_(H)17 and Treg cell function may be central in some of these diseases.

Although many studies have analyzed the role of T_(H)17 cells in animalmodels of intestinal inflammation and autoimmunity, there are only a fewstudies investigating the role of T_(H)17 cells in patients with Crohn'sdisease. An increased number of T cells are found expressing retinoidrelated orphan receptor-ct (RORγt), the master transcription factor forT_(H)17 cells, in the lamina propria of patients with Crohn's disease.Two independent studies showed that T_(H)17 cells in human peripheralblood and in the gut from healthy individuals and patients with Crohn'sdisease (Acosta-Rodriguez et al., 2007; Annunziato et al., 2007). Thesetwo studies showed that these cells are characterized by the expressionof RORγt, IL23R and CCR6, whereas they lack CXCR3, a chemokine receptorthat is characteristic for T_(H)1 cells.

The study by Annunziato et al. (2007) demonstrated IL-17A-producing Tcells in the gut, including T cell populations which also expressed bothIL17A and IFNγ, which they named “T_(H)17/T_(H)1” cells.Acosta-Rodriguez et al. (2007) identified T_(H)17 cells that can becharacterized by CCR6⁺CCR4⁺ expression, while CCR6⁺CXCR3⁺-expressingT_(H)1 cells also included a subset which produced both IL17A and IFNγ.Moreover, very recent findings implicate CD161 as a novel surface markerfor human T_(H)17 cells and demonstrate the exclusive origin of thesecells from a CD161⁺CD4⁺ T cell progenitor. The interactions betweenT_(H)1 and T_(H)17 cells and the role of IFNγ on T_(H)17 cells may bemore complex than previously assumed and require further analysis todelineate the specific contributions of these cell lineages to Crohn'sdisease and other autoimmune diseases.

IL-6, a protein encoded by the IL6 gene, is an interleukin that actsboth as a pro-inflammatory and an anti-inflammatory cytokine. It issecreted by T cells and macrophages to stimulate immune response, e.g.during infection and after trauma, IL-6's role as an anti-inflammatorycytokine is mediated through its inhibitory effects on TNF-alpha andIL-1, and activation of IL-1ra and IL-10.

IL-23 is a heterodimeric cytokine consisting of two subunits, one calledp40, which is shared with another cytokine, IL-12, and another calledp19 (the IL-23 alpha subunit which is encoded by the IL-23A gene) (seeFIG. 10A). The two subunits of IL-23 are linked by a disulfide bridge.IL-23 is an important part of the inflammatory response againstinfection. It promotes upregulation of the matrix metalloprotease MMP9,increases angiogenesis and reduces CD8+T-cell infiltration.

Crohn's disease and ulcerative colitis are the two main disease entitiesof inflammatory bowel diseases (IBDs). Crohn's disease has an averageannual incidence rate of 6.3 per 100,000 people in the US. Althoughtheir exact aetiology is still not completely understood, it has beenproposed that their pathogenesis is characterized by an exaggeratedimmune response in genetically susceptible individuals. For many yearsit has been assumed that Crohn's disease is mainly mediated by T_(H)1cells, while ulcerative colitis is a T_(H)2-like type of inflammation.This has been supported by increased levels of T_(H)1 cytokines such asIFNγ and interleukin 12 (IL-12) in Crohn's disease and an increasedexpression of certain T_(H)2 cytokines such as IL-13 in ulcerativecolitis.

Ustekinumab (CNTO 1275; Stelara™; Centocor, Inc., Malvern, Pa.) is ahuman, immunoglobulin G1 kappa (IgG1κ) monoclonal antibody thatspecifically binds the shared p40 subunit of IL-12 and IL-23 andinhibits the interactions of IL-12 and IL-23 with the cell surfaceIL-12Rβ1 receptor, thus preventing IL-12- or IL-23-mediated signalingcascades.

Tocilizumab (Actemra) is a humanized recombinant IgG1k monoclonalantibody against the IL-6 receptor. Tocilizumab was approved by the FDAon Jan. 8, 2010 for the treatment of rheumatoid arthritis.

However, there remains a need for further effective therapies that treatdiseases in which T_(H)17 and T_(H)22 cell mediated responses play arole. Furthermore given the complexity of the immunological responsesinvolved in these diseases there is a need for therapies that act onmultiple pathways (e.g. T_(H)17 and T_(H)1),

Providing such therapies in the form of a bispecific construct (e.g. onethat is specific for IL-6 and IL-23 (and optionally IL-12 as well)represents a significant challenge. Such bivalent bispecific constructsneed not only specific antigen binding and neutralizing domains, butalso to be stable, have a long mean residence time and efficacy in vivo.While many efforts have been made to create bispecific antibodies, allefforts to date have failed to create such stable molecules with long invivo residence times. Moreover, the many efforts to create bispecificsthrough genetic fusion methods have not succeeded in creating readilymanufactured molecules that are stable and high affinity. The bispecificconstructs described herein solve these problems for the first time.

Bispecific antibodies targeting T_(H)17 cells have been developed bytargeting IL23 and IL17A, (Mabry R. et al., 2009).

The inventors aim to provide useful bispecific antibodies.

The inventors' novel approach uses highly efficient production of scFvin prokaryotic systems, and a site-specific chemical conjugation methodto generate large quantities of bispecific proteins, avoiding many ofthe problems that plague alternative methods of bispecific antibodygeneration. A key step is the use of a flexible linker that ischemically attached to the polypeptide chains, prior to refolding, thatallow each scFv to refold independently of the other to lead to afunctional bispecific construct.

The inventors method involves use of in vivo site specific incorporationof non natural aminoacids functioning as reactive sites for covalent andsite specific binding of a linker, such as PEG, to the target protein(see WO 2007/130453, the entire contents of which are hereinincorporated by reference).

An advantage of the method is that the chemistry used to conjugate scFvsto the linker is orthogonal to the 20 natural amino acids.

Another method of incorporating non-natural amino acids intopolypeptides is described in U.S. Pat. No. 7,632,024 (Cho et al).

According to the present invention, single-chain variable fragments(scFv) are readily produced in large quantities and can be easilypurified. B-cell cloning, and rescue of rabbit antigen specificmonoclonal antibodies, following functional screens, permits theidentification of high quality antibodies. The antibodies aresubsequently humanized and converted to scFv.

The Inventors have identified a number of humanized monoclonalantibodies for specific targets, namely human IL-6, human IL-23 andhuman IL-12.

Furthermore, they have generated antibody fragments and engineered themin order to generate bispecific ScFv molecules targeting IL-6 and IL-23or IL-6 and IL12/23 to be used in therapy where inhibition of T_(H)1and/or T_(H)17 cells is beneficial, including inflammatory andautoimmune diseases.

Aliahmadi et al (2009) Eur J Immunol 39, 1221-1230 describes certainexperiments involving a combination of a goat polyclonal serum againstisolated human IL-23 p19 subunit and an anti-IL-6 monoclonal antibody.

SUMMARY OF THE INVENTION

The present invention provides bivalent, bispecific constructscomprising an anti-IL-6 antibody, or derivative thereof, and ananti-IL-23 antibody, or derivative thereof, methods of making suchconstructs, and use of such constructs in therapy.

The antibodies of the bivalent, bispecific constructs of the presentinvention may be isolated monoclonal antibodies, preferably, they areisolated human monoclonal antibodies.

The antibodies of the bivalent, bispecific constructs of the presentinvention may be chimeric antibodies. In a preferred embodiment theframework regions of the antibodies, or derivatives thereof, of thepresent invention have been humanized.

The antibody derivatives of the present invention may include the entirevariable region, the heavy chain of the variable region (VH), the lightchain of the variable region (VL), a Fab, a Fab′, a F(ab′)2, a Fv, ascFv, a dAb or a complementarity determining region (CDR). The antibodyderivatives of the present invention entirely retain, or substantiallyretain, the antigen binding activity of the antibodies from which theyare derived. In a preferred embodiment the antibody derivatives arescFv.

The present invention further provides an anti-IL-6 antibody, orderivative thereof, and an anti-IL-23 antibody, or derivative thereof,methods of making such antibodies, or derivatives thereof and use ofsuch such antibodies, or derivatives thereof alone or in combination intherapy

The antibodies and antibody derivatives of the present invention(including bispecific constructs) may be modified to incorporate one ormore non-natural amino acids.

In an embodiment the anti-IL-6 antibody, or derivative thereof, maycomprise particular motifs from the CDR regions of the 13A8 antibody. Assuch the present invention provides an anti-IL-6 antibody, or derivativethereof, which comprises:

a CDR2 region comprising the amino acid sequence YIYTDX¹STX²YANWAKG,whereinX¹ is selected from the group consisting of glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine; andX² is selected from the group consisting of phenylalanine, tryptophan,and tyrosine; andpreferably X¹ is serine or threonine and X2 is tryptophan or tyrosine;(SEQ ID NO. 335) and/ora CDR5 region comprising the amino acid sequence RX¹STLX²S, wherein X¹and X² are independently alanine or threonine. (SEQ ID NO. 336)

In an embodiment, the anti-IL-6 antibody, or derivative thereof, maycomprise at least one, at least two, at least three, at least four, atleast five or six CDR regions whose amino acid sequence is selected fromthe group consisting of SEQ ID NOs. 10-15. The CDR region may beselected from the CDRs of the heavy chain of the variable region (VH)(i.e. SEQ ID NOs. 10-12) and/or from the CDRs of the light chain of thevariable region (VL) (i.e. SEQ ID NOs 13-15). In a particular embodimentthe anti-IL-6 antibody, or derivative thereof, may comprise all of theamino acid sequences of SEQ ID NOs 10-15.

The anti-IL-6 antibody, or derivative thereof, may comprise the entireVH and/or VL of an anti-IL-6 antibody, or derivative thereof. In aparticular embodiment the anti-IL-6 antibody, or derivative thereof, maythe comprise the VH of an anti-IL-6 antibody, the VH having the sequenceof SEQ ID NO. 259 and/or the VL of an anti-IL-6 antibody, the VL havingthe sequence of SEQ ID NO. 261.

In a preferred embodiment the bivalent, bispecific construct comprisesan anti-IL-6 antibody, or derivative thereof, which is a scFv comprisinga heavy chain comprising at least one, at least two or three CDR regionsthe amino acid sequence of SEQ ID NO. 10-12 and a light chain comprisingat least one, at least two or three CDR regions the amino acid sequenceof SEQ ID NOs. 13-15. In an embodiment the scFv may comprise the aminoacid sequence of SEQ ID NO. 259 and a light chain comprising the aminoacid sequence of SEQ ID NO. 261.

The invention also provides an anti-IL-6 antibody, or derivativethereof, or a bivalent bispecific construct comprises an anti-IL-6antibody, or derivative thereof according to the above clauses based onantibodies 28D2, 18D4, 8C8, 9H4 and 9C8 in which reference to SEQ ID NOs10-15 is replaced by reference, respectively, to SEQ ID NOs 20-25,30-35, 40-45, 50-55 and 60-65. For anti-IL-6 antibodies and derivativesbased on 28D2, reference to SEQ ID NOs. 259 and 261 may be replaced byreferences to SEQ ID NOs. 263 and 265.

The present invention also encompasses bivalent, bispecific constructshaving anti-IL-6 antibodies, or derivatives thereof, which comprise atleast one CDR region whose amino acid sequence has at least 90%, atleast 95%, at least 98%, or at least 99% identity to an amino acidsequence selected from the group consisting of SEQ ID NO.s 10-15.Similarly, the present invention also encompasses bivalent, bispecificconstructs having anti-IL-6 antibodies, or derivatives thereof, whichcomprise at least one CDR region whose amino acid sequence comprises oneor more amino acid additions, deletions or substitutions to an aminoacid sequence selected from the group consisting of SEQ ID NO.s 10-15.In an embodiment the CDR region comprises at least one conservativeamino acid substitution to an amino acid sequence selected from thegroup consisting of SEQ ID NO.s 10-15.

The present invention also provides bivalent, bispecific constructcomprising an anti-IL-6 antibody, or derivative thereof, which comprisesat least one CDR region that binds to the same epitope as an anti-IL-6antibody having CDRs corresponding to the amino acid sequences of SEQ IDNO.s 10-15.

In an embodiment the anti-IL-6 antibody, or derivative thereof, isselected from, or derived from, the group consisting of 13A8, 9H4, 9C8,8C8, 18D4 and 28D2.

In another embodiment the anti-IL-23 antibody, or derivative thereof,may comprise particular motifs from the CDR regions of the 31A12antibody. As such the present invention provides an anti-IL-23 antibody,or derivative thereof, which comprises:

a CDR2 region comprising the amino acid sequence YYAX¹WAX²G, whereinX′ is selected from the group consisting of serine, proline andaspartate, andX² is selected from the group consisting of lysine and glutamine; (SEQID 337) and/ora CDR5 region comprising the amino acid sequence AX¹TLX²S, whereinX¹ is selected from the group consisting of serine and alanineX² is selected from the group consisting of alanine and threonine. (SEQID 338)

As used herein, CDR1 refers to VH CDR1, CDR2 refers to VH CDR2, CDR3refers to VH CDR3, CDR4 refers to VL CDR1, CDR5 refers to VL CDR2 andCDR6 refers to VL CDR3.

In another embodiment, the anti-IL-23 antibody, or derivative thereof,may comprise at least one, at least two, at least three, at least four,at least five or six CDR regions whose amino acid sequences are selectedfrom the group consisting of SEQ ID NOs 90-95. The CDR region may beselected from the CDRs of the heavy chain of the variable region (VH)(i.e. SEQ ID NOs. 90-92 and/or from the CDRs of the light chain of thevariable region (VL) (i.e. SEQ ID NO. 93-95). In a particular embodimentthe anti-IL-23 antibody, or derivative thereof, may comprise all of theamino acid sequences of SEQ ID NO.s 90-95.

The anti-IL-23 antibody, or derivative thereof, may comprise the entireVH and/or VL of an anti-IL-23 antibody or derivative thereof. In aparticular embodiment the anti-IL-23 antibody, or derivative thereof,may comprise the VH of an anti-IL-23 antibody, the VH having thesequence of SEQ ID NO. 267 and/or the VL of an anti-IL-23 antibody, theVL having the sequence of SEQ ID NO. 269.

In a preferred embodiment the bivalent, bispecific construct comprisesan anti-IL-23 antibody, or derivative thereof, which is a scFvcomprising a heavy chain comprising

at least one, at least two or three CDR regions having the amino acidsequence of SEQ ID NO. 90-92 and a light chain comprising at least one,at least two or three CDR regions having the amino acid sequence of SEQID NOs. 93-95. In an embodiment the scFv may comprise the amino acidsequence of SEQ ID NO. 267 and a light chain comprising the amino acidsequence of SEQ ID NO. 269.

The invention also provides an anti-IL-23 antibody, or derivativethereof, or a bivalent bispecific construct comprises an anti-IL-23antibody, or derivative thereof according to the above clauses based onantibodies 49B7, 16C6, 34E11 and 35H4 in which reference to SEQ ID NOs90-95 is replaced by reference, respectively, to SEQ ID Nos 100-105,110-115, 120-25 and 130-135.

The present invention also encompasses bivalent, bispecific constructshaving anti-IL-23 antibodies, or derivatives thereof, which comprise atleast one CDR region whose amino acid sequence has at least 90%, atleast 95%, at least 98%, or at least 99% identity to an amino acidsequence selected from the group consisting of SEQ ID NO.s 90-95.Similarly, the present invention also encompasses bivalent, bispecificconstructs comprising an anti-IL-23 antibody, or derivative thereof,which comprise at least one CDR region whose amino acid sequencecomprises one or more amino acid additions, deletions or substitutionsto an amino acid sequence selected from the group consisting of SEQ IDNOs. 90-95. In an embodiment the CDR region comprises at least oneconservative amino acid substitution to an amino acid sequence selectedfrom the group consisting of SEQ ID NO.s 90-95.

The present invention also provides bivalent, bispecific constructcomprising an anti-IL-23 antibody, or derivative thereof, whichcomprises at least one CDR region that binds to the same epitope as ananti-IL-6 antibody having CDRs corresponding to the amino acid sequencesof SEQ ID NO.s 90-95.

In an embodiment the anti-IL-23 antibody, or derivative thereof, isselected from, or derived from, the group consisting of 31A12, 34E11,35H4, 49B7 and 16C6. It is likely that such antibodies bind to the p19subunit of IL-23.

In another embodiment the anti-IL-23 antibody, or derivative thereof,may also bind IL-12. Without being bound by theory it is likely thatsuch antibodies bind to the p40 subunit that is shared by both IL-23 andIL-12. Such antibodies are referred to herein as anti-IL-23/IL-12antibodies.

It is not excluded that antibodies bind to p40 and inhibit IL-23 yet doinhibit IL-12—such antibodies are included within the scope of“anti-IL-23 antibodies”.

In an embodiment the present invention provides anti_IL-23/IL-12antibodies, or derivatives thereof, which may comprise particular motifsfrom the CDR regions of the 45G5 or 22H8 antibodies. As such the presentinvention provides an anti-IL-23/IL-12 antibody, or derivative thereof,which inhibits both IL-12 and IL-23 which comprises:

a CDR2 region comprising the amino acid sequence sequence WX¹KG, whereinX1 is alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine or tryptophan, and preferably is alanine or valine (SEQ IDNO. 358);and/ora CDR3 region comprising the amino acid sequence YAYX¹GDAFDP, wherein X¹is alanine or isoleucine; (SEQ ID NO. 339)and/ora CDR3 region comprising the amino acid sequence SDYFNX¹, wherein X¹ isisoleucine or valine; (SEQ ID NO. 340)and/ora CDR4 region comprising the amino acid sequence QX¹SQX², whereinX¹ is alanine or serine, andX² is selected from the group consisting of glycine, asparagine,glutamine, cysteine, serine, threonine, and tyrosine;preferably X² is serine or threonine;and/or (SEQ ID NO. 359)a CDR5 region comprising the amino acid sequence ASX¹LA, wherein X¹ islysine or threonine. (SEQ ID NO. 341)and/ora CDR6 region comprising the amino acid sequence QSYYDX¹NAGYG, whereinX¹ is alanine or valine. (SEQ ID NO. 342)

In an embodiment the anti-IL-23/IL-12 antibody or derivative thereof maycomprise at least one, at least two, at least three, at least four, atleast five or six CDR regions whose amino acid sequences are selectedfrom the group consisting of SEQ ID NO.s 140-145. The CDR region may beselected from the CDRs of the heavy chain of the variable region (VH)(i.e. SEQ ID NOs. 140-142 and/or from the CDRs of the light chain of thevariable region (VL) (i.e. SEQ ID NOs. 143-145. In a particularembodiment the anti-IL-23/IL-12 antibody, or derivative thereof, maycomprise all of the amino acid sequences of SEQ ID NO.s 140-145.

The anti-IL-23/IL-12 antibody, or derivative thereof, may comprise theentire VH and/or VL of an anti-IL-23/IL-12 antibody or derivativethereof. In a particular embodiment the anti-IL-23/IL-12 antibody, orderivative thereof, may comprise the VH of an anti-IL-23/IL-12 antibody,the VH having SEQ ID NO. 271 and/or the VL of an anti-IL-23/IL-12antibody, the VL having SEQ ID NO. 273.

In a preferred embodiment the bivalent, bispecific construct comprisesan anti-IL-23/IL-12 antibody, or derivative thereof, which is a scFvcomprising at least one, at least two or three CDR regions having theamino acid sequence of SEQ ID NOs. 140-142 and a light chain comprisingat least one, at least two or three CDR regions having the amino acidsequence of SEQ ID NOs. 143-145. In an embodiment the scFv may comprisea heavy chain comprising the amino acid sequence of SEQ ID NO. 271 and alight chain comprising the amino acid sequence of SEQ ID NO. 273.

The invention also provides an anti-IL-23/IL-12 antibody, or derivativethereof, or a bivalent bispecific construct comprises ananti-IL-23/IL-12 antibody, or derivative thereof according to the aboveclauses based on antibodies 45G5, 1H1, 4F3, 5C5 and 14B5 in whichreference to SEQ ID NOs 140-145 is replaced by reference, respectively,to SEQ ID NOs 150-155, 160-165, 170-175, 180-185 and 190-195. Foranti-IL-23/IL-12 antibodies and derivatives based on 45G5, reference toSEQ ID NOs. 271 and 273 may be replaced by references to SEQ ID NOs. 275and 277.

The present invention also encompasses bivalent, bispecific constructscomprising anti-IL-23/IL-12 antibodies, or derivatives thereof, whichcomprise at least one CDR region whose amino acid sequence has at least90%, at least 95%, at least 98%, or at least 99% identity to an aminoacid sequence selected from the group consisting of SEQ ID NO.s 140-145.Similarly, the present invention also encompasses bivalent, bispecificconstructs comprising anti-IL-23/IL-12 antibodies, or derivativesthereof, which comprise at least one CDR region whose amino acidsequence comprises one or more amino acid additions, deletions orsubstitutions to an amino acid sequence selected from the groupconsisting of SEQ ID NO.s 140-145. In an embodiment the CDR regioncomprises at least one conservative amino acid substitution to an aminoacid sequence selected from the group consisting of SEQ ID NO.s 140-145.

The present invention also provides bivalent, bispecific constructhaving an anti-IL-23/IL-12 antibody, or derivative thereof, whichcomprises at least one CDR region that binds to the same epitope as ananti-IL-23/IL-12 antibody having CDRs corresponding to the amino acidsequences of SEQ ID NOs. 140-145. In an embodiment the epitope ispresent on the p40 subunit that is common to both IL-12 and IL-23.

In an embodiment the anti-IL-23/IL-12 antibody, or derivative thereof,is selected from, or derived from, the group consisting of 22H8, 45G5,14B5, 4F3, 5C5, and 1H1.

It will be appreciated that the anti-IL-6 antibodies and derivativesthereof, described above that may form part of a bivalent bispecificconstruct may be independently combined with the anti-IL-23 antibodies,or derivatives thereof, (including the anti-IL-23/IL-12 antibodies, andderivatives thereof) described above in a single bivalent bispecificconstruct. In such constructs, both the anti-IL-6 antibody, orderivative thereof, and the anti-IL-23 antibody, or derivative thereof,may incorporate non-natural amino acids, through which the anti-IL-6antibody, or derivative thereof, is coupled to the anti-IL-23 antibody,or derivative thereof.

The bivalent bispecific constructs of the present invention may furthercomprise a linker between a non-natural amino acid in each antibody, orderivative thereof. The bivalent bispecific constructs of the presentinvention may further comprise polyethylene glycol molecules (PEG). ThePEG molecule may optionally serve as a linker between the anti-IL-6antibody, or derivative thereof, and the anti-IL-23 antibody, orderivative thereof, (including anti-IL-23/IL-12 antibodies, orderivatives thereof). Suitably, other water soluble polymers, such aspolyvinylalcohol, polysaccharides, polyalkylene oxides, hydroxyethylstarch, and polyols, may also be used.

The anti-IL-6 and anti-IL-23 antibodies or derivatives thereof,(including anti-IL-23/IL-12 antibodies, or derivatives thereof)described herein are useful per se. In another aspect of the inventionthe present invention provides anti-IL-6 and anti-IL-23 antibodies orderivatives thereof, (including anti-IL-23/IL-12 antibodies, orderivatives thereof), that have particular utility in the manufacture ofbivalent bispecific constructs of the invention and/or in combinationtherapeutics. Said anti-IL-6 and anti-IL-23 or derivatives thereof,(including anti-IL-23/IL-12 antibodies, or derivatives thereof) mayoptionally be modified to increase half life (for instance throughPEGylation). Anti-IL-6 antibodies, or derivatives thereof, andanti-IL-23 antibodies, or derivatives thereof, may incorporatenon-natural amino acids to facilitate linkage of PEG groups.

An aspect of the invention provides a combination (for separate,sequential or separate administration) comprising an anti-IL-6 antibodyor derivative thereof and an anti-IL-23 antibody or derivative thereof(which may, for example, by an anti-IL-23/IL-12 antibody or derivativethereof).

The present invention further encompasses bivalent bispecific constructscomprising anti-IL-6 and anti-IL-23 antibody derivatives (includinganti-IL-23/IL-12 antibody derivatives) wherein said antibody derivativesare selected from Fab, Fab′, F(ab)′, Single Chain Antibodies (scFv),kappabodies, Minibodies and Janusins.

As such the present invention provides the following antibodies orderivatives thereof:

An anti-IL-6 antibody, or derivative thereof, which comprises a heavychain comprising the amino sequence of SEQ ID NO. 259 and a light chaincomprising the amino sequence of SEQ ID NO. 261.

An anti-IL-23 antibody or derivative thereof, which comprises a heavychain comprising the amino sequence of SEQ ID NO. 267 and a light chaincomprising the amino sequence of SEQ ID NO. 269.

An anti-IL-23/IL-12 antibody, or derivative thereof, which comprises aheavy chain comprising the amino sequence of SEQ ID NO. 271 and a lightchain comprising the amino sequence of SEQ ID NO. 273.

In another aspect of the present invention a polynucleotide encoding aportion of a bivalent, bispecific construct of the present invention isprovided. Such polynucleotides may encode an antibody, or derivativethereof, as disclosed herein

The present invention also provides vectors comprising suchpolynucleotides, host cell comprising such vectors (optionally the hostcells are auxotrophic), oligonucleotide primers for cloning andexpressing antibodies, or derivative thereof, as disclosed herein.Particular oligonucleotide primer of the present invention includeoligonucleotide primers comprising one of the nucleotide sequences setout in any one of 200-258.

In another aspect of the invention methods for producing a bivalent,bispecific construct is provided. The method may comprise:

-   -   (a) providing an anti-IL-6 antibody, or derivative thereof        modified by the incorporation of at least one non-natural amino        acid;    -   (b) providing an anti-IL-23 antibody, or derivative thereof        modified by the incorporation of at least one non-natural amino        acid;    -   (c) reacting the modified anti-IL-6 antibody, or modified        derivative thereof, with the modified anti-IL-23 antibody, or        modified derivative thereof, such that the two are coupled        through a linkage between a non-natural amino acid of each        portion.

The method may comprise coupling the modified anti-IL-6 antibody, ormodified derivative thereof, and the modified anti-IL-23 antibody, ormodified derivative thereof, through a linkage comprising a linkerportion, wherein one end of the linker portion is coupled to anon-natural amino acid of the modified anti-IL-6 antibody, or modifiedderivative thereof, and the other end of the linker portion is coupledto a non-natural amino acid of modified anti-IL-23 antibody, or modifiedderivative thereof. Examples of suitable linkers are known in the artand include short peptide sequences. The present invention also providesfor the use of PEG as a linker. Thus, in an embodiment the linkerportion may be a PEG molecule.

The method may comprise the use of non-natural amino acids that containa group selected from:

an azide, cyano, nitrile oxides, alkyne, alkene, strained cyclooctyne,strained cycloalkene, cyclopropene, norbornenes or aryl, alkyl or vinylhalide, ketone, aldehyde, ketals, acetals hydrazine, hydrazide, alkoxyamine, boronic acid, organotin, organosilicon, beta-silyl alkenylhalide, beta-silyl alkenyl sulfonates, pyrones, tetrazine, pyridazine,aryl sulfonates, thiosemicarbazide, semicarbazide, tetrazole,alpha-ketoacid group prior to linkage to the other portion. Inparticular the non-natural amino acid may be azidohomoalanine,homopropargylglycine, homoallylglycine, p-bromophenylalanine,p-iodophenylalanine, azidophenylalanine, acetylphenylalanine orethynylephenylalanine, amino acids containing an internal alkene such astrans-crotylalkene, serine allyl ether, allyl glycine, propargylglycine, vinyl glycine, pyrrolysine,N-sigma-o-azidobenzyloxycarbonyl-L-Lysine (AzZLys),N-sigma-propargyloxycarbonyl-L-Lysine,N-sigma-2-azidoethoxycarbonyl-L-Lysine,N-sigma-tert-butyloxycarbonyl-L-Lysine (BocLys),N-sigma-allyloxycarbonyl-L-Lysine (AlocLys), N-sigma-acetyl-L-Lysine(AcLys), N-sigma-benzyloxycarbonyl-L-Lysine (ZLys),N-sigma-cyclopentyloxycarbonyl-L-Lysine (CycLys),N-sigma-D-prolyl-L-Lysine, N-sigma-nicotinoyl-L-Lysine (NicLys),N-sigma-N-Me-anthraniloyl-L-Lysine (NmaLys), N-sigma-biotinyl-L-Lysine,N-sigma-9-fluorenylmethoxycarbonyl-L-Lysine, N-sigma-methyl-L-Lysine,N-sigma-dimethyl-L-Lysine, N-sigma-trimethyl-L-Lysine,N-sigma-isopropyl-L-Lysine, N-sigma-dansyl-L-Lysine,N-sigma-o,p-dinitrophenyl-L-Lysine, N-sigma-p-toluenesulfonyl-L-Lysine,N-sigma-DL-2-amino-2carboxyethyl-L-Lysine,N-sigma-phenylpyruvamide-L-Lysine, N-sigma-pyruvamide-L-Lysine; andparticularly a group selected from:an azide, alkyne, alkene, or aryl, alkyl or vinyl halide, ketone,aldehyde, hydrazine, hydrazide, alkoxy amine, boronic acid, organotin,organosilicon group prior to linkage to the other portion. In particularthe non-natural amino acid may be azidohomoalanine,homopropargylglycine, homoallylglycine, p-bromophenylalanine,p-iodophenylalanine, azidophenylalanine, acetylphenylalanine orethynylephenylalanine, amino acids containing an internal alkene such astrans-crotylalkene, serine allyl ether, allyl glycine, propargylglycine, vinyl glycine.

The method may comprise coupling the modified anti-IL-6 antibody, ormodified derivative thereof, and the modified anti-IL-23 antibody, ormodified derivative thereof using a [3+2] cycloaddition/[3+2] dipolarcycloaddition or azide-alkyne cycloaddition reaction commonly referredto as Click reaction (which may be catalyzed by copper(I), ruthenium,other metals, or promoted by strain and/or electron withdrawing groups),a Heck reaction, a Sonogashira reaction, a Suzuki reaction, a Stillecoupling, a Hiyama/Denmark reaction, olefin metathesis, a Diels-alderreaction, carbonyl condensation with hydrazine, hydrazide, alkoxy amineor hydroxyl amine. A Staudinger ligation is also possible.

The method may also comprise:

-   -   (a) providing a host cell, the host cell comprising a vector        having a polynucleotide encoding an anti-IL-6 antibody, or        derivative thereof, which anti-IL-6 antibody, or derivative        thereof, is modified by the incorporation of at least one        non-natural amino acid;    -   (b) providing a host cell, the host cell comprising a vector        having a polynucleotide encoding an anti-IL-23 antibody, or        derivative thereof, which anti-IL-23 antibody, or derivative        thereof, is modified by the incorporation of at least one        non-natural amino acid;    -   (c) growing the host cells under conditions such that the host        cells express the modified anti-IL-6 antibody, or derivative        thereof, or the modified anti-IL-23 antibody, or derivative        thereof,    -   (d) isolating the anti-IL-6 antibody, or derivative thereof, and        the anti-IL-23 antibody, or derivative thereof;    -   (e) reacting the modified anti-IL-6 antibody, or derivative        thereof, with the modified anti-IL-23 antibody, or derivative        thereof, such that the modified anti-IL-6 antibody, or        derivative thereof, is coupled to the modified anti-IL-23        antibody, or derivative thereof, through a linkage between a        non-natural amino acid of each modified antibody, or derivative        thereof.

As discussed in more detail below, the method may also compriseincorporating a non-natural amino acid (e.g. Aha) by incorporating it ata specific selected amino acid encoded position (typically a methionineencoded position), and if necessary mutating the polynucleotide sequenceof the target protein to eliminate methionine (or other specificselected amino acid) codons at positions in which it is not desired toincorporate a non-natural amino acid and/or if necessary mutating thepolynucleotide sequence of the target protein to provide one or more(typically one) new methionine (or other specific selected amino acid)codons at positions in which it is desired to incorporate a non-naturalamino acid.

In another aspect of the invention a method of selecting parentantibodies suitable for inclusion in a bivalent, bispecific construct ofthe present invention, comprising the steps of:

(i) selecting B cells specific for IL-6 or IL-23;(ii) aliquoting out separate samples of the B-cells (e.g. into the wellsof a 96 cell well plate);(iii) culturing the B cells;(iv) separately harvesting the supernatant, which contains theantibodies, from each aliquoted sample;(v) assaying the supernatant from each aliquoted sample for IL-6 orIL-23 binding (e.g. using an ELISA);(vi) assaying the supernatant from each aliquoted sample for inhibitionof IL-6 or 11-23 activity;(vii) selecting the antibodies from the wells that showed high levels ofinhibition of IL-6 or IL-23 activity and/or strong IL-6 or IL-23binding; and(viii) optionally assaying the supernatant from the IL-23 aliquotedsamples for inhibition of IL-12 activity; and(ix) selecting IL-23 antibodies that additionally show high levels ofIL-12 activity and/or strong IL-12 binding as parent antibodies.

In another aspect of the invention, the bivalent, bispecific constructas described above is provided for use in therapy. Generally, thebivalent, bispecific construct described above for use in treating aT_(H)17, T_(H)22 and/or T_(H)17 and T_(H)1 mediated disease by bindingone or more molecules involved in the differentiation of T_(H)17 cellsor binding one or more molecules produced by activated T_(H)17 cells. Ina particular embodiment the such diseases include multiple sclerosis,psoriasis, psoriatic arthritis, pemphigus vulgaris, organ transplantrejection, Crohn's disease, inflammatory bowel disease (IBD), irritablebowel syndrome (IBS), lupus erythematosis, and diabetes.

The present invention also provides a combination therapeutic comprisingan anti-IL-6 antibody and an anti-IL-23 antibody, for use in treating aT_(H)17, T_(H)22 and/or T_(H)17 and T_(H)1 mediated disease. Inparticular such combinations are provided for use the treatment ofinflammatory and autoimmune disorders.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a B cell selection. Each well from one 96 well plate of Bcells was assayed for both IL-6 neutralization by the B9 cell lineproliferation assay and IL-6 binding by ELISA. The results from eachassay are aligned for comparison.

FIG. 2 shows an outline of the experimental process from V-region rescueto scFv generation

FIGS. 3A and B show the human and primate IL-6 neutralization activityof selected anti-IL6 rabbit/human chimeric antibodies: Rabbit/humanchimeric mAbs were expressed in mammalian cells. The mAbs werequantitated in the supernatants by ELISA. They were tested forneutralization of 50 pg/ml of human IL-6 or 100 pg/ml of primate IL-6,as indicated, using the B9 cell proliferation assay.

FIGS. 3C and D show the human and primate IL-6 neutralization activityof selected anti-IL6 rabbit/human chimeric antibodies: Rabbit/humanchimeric mAbs were expressed in mammalian cells. The mAbs werequantitated in the supernatants by ELISA. They were tested forneutralization of 50 pg/ml of human IL-6 or 100 pg/ml of primate IL-6,as indicated, using the B9 cell proliferation assay.

FIG. 3E shows the human IL-6 neutralization activity of selectedanti-IL6 rabbit/human chimeric antibodies: Rabbit/human chimeric mAbswere expressed in mammalian cells. The mAbs were quantitated in thesupernatants by ELISA. They were tested for neutralization of 50 pg/mlof human IL-6 or 100 pg/ml of primate IL-6, as indicated, using the B9cell proliferation assay.

FIG. 4 shows B cell selection Each well from one B cells 96-well platewas assayed for IL-23 neutralization and IL-23 binding. The results ofthe two assays for each well are aligned for comparison.

FIGS. 5A-5I show human IL-23 neutralization activity of selectedanti-IL-23 rabbit/human chimeric antibodies. Candidate mAbs were derivedand tested for neutralization of heterodimeric recombinant IL-23 (eBioIL23)

FIGS. 6A and 6B show the neutralization activity of primate and humanIL-23 Transfection supernatants of several mAbs were compared forneutralization of either heterodimeric human (panel B) or primate IL-23(panel A) using the mouse splenocyte assay. Ig levels were measured inthe transfection supernatants to allow comparison of specificactivities.

FIG. 7A shows the structure of IL-12 and IL-23

FIG. 7B shows the IL-12 and IL-23 Receptors and their associatedmechanisms.

FIG. 8A shows transfection supernatants of several mAbs were comparedfor neutralization of human IL-12 using the NK92 cell line assay.

FIG. 8B shows neutralization of Human IL-23 by selected mAbs mAbs thatwere positive in the primary rescue transfections from B cell clones,were expressed in HEK293 transient transfections, in which IgGconcentration was quantitated for calculating EC50 values. The mAbs weretested for neutralization of 1200 pg/ml of IL-23 (eBiosciences).

FIG. 78C shows neutralization of Human IL-23 by selected mAbs mAbs thatwere positive in the primary rescue transfections from B cell clones,were expressed in HEK293 transient transfections, in which IgGconcentration was quantitated for calculating EC50 values. The mAbs weretested for neutralization of 1200 pg/ml of IL-23 (eBiosciences).

FIG. 8D shows neutralization of Human IL-23 by selected mAbs mAbs thatwere positive in the primary rescue transfections from B cell clones,were expressed in HEK293 transient transfections, in which IgGconcentration was quantitated for calculating EC50 values. The mAbs weretested for neutralization of 1200 pg/ml of IL-23 (eBiosciences).

FIG. 8E shows neutralization of Human IL-23 by selected mAbs mAbs thatwere positive in the primary rescue transfections from B cell clones,were expressed in HEK293 transient transfections, in which IgGconcentration was quantitated for calculating EC50 values. The mAbs weretested for neutralization of 1200 pg/ml of IL-23 (eBiosciences).

FIGS. 8F-8G show neutralization of Human IL-12 by selected mAbs mAbsthat were positive in the primary rescue transfections from B cellclones, were expressed in HEK293 transient transfections, in which IgGconcentration was quantitated for calculating EC50 values. The mAbs weretested for neutralization of 1000 pg/ml of human IL-12.

FIGS. 8H and 8I show neutralization of Primate IL-23 by selected mAbsmAbs that were positive in the primary rescue transfections from B cellclones, were expressed in HEK293 transient transfections, in which IgGconcentration was quantitated for calculating EC50 values. The mAbs weretested for neutralization of 1000 pg/ml of primate IL-23.

FIG. 9A shows Rabbit scFvs were expressed in mammalian cells. The scFvswere quantitated in the supernatants by SDS PAGE. They were tested forneutralization of 200 pg/ml of human IL-6 as indicated, using the B9cell proliferation assay.

FIG. 9B shows 9C8 humanization. 9C8, a high affinity and high potencychimeric mAb, was humanized by changing the framework regions of the VHand VL to human framework sequences, with limited back mutation torabbit framework sequences. Different human framework sequences werecompared for humanization of 9C8. The humanized mAbs were expressed bytransient transfection of HEK293 cells. Transfection supernatants weretested for the ability to neutralize 50 pg/ml of human IL-6 using the B9cell proliferation assay.

FIG. 9C shows Humanized Anti-IL-6 mAbs. 9C8 and 18D4, high affinity andhigh potency chimeric mAbs, were humanized by changing the frameworkregions of the VH and VL to human framework sequences, with limited backmutation to rabbit framework sequences. The humanized mAbs wereexpressed by transient transfection of HEK293 cells. IgG in thetransfection supernatants were quantitated and they were tested for theability to neutralize 50 pg/ml of human IL-6 using the B9 cellproliferation assay.

FIG. 9D shows Humanized Anti-IL-6 mAbs and scFvs. 9C8, a high affinityand high potency chimeric mAb, was humanized by changing the frameworkregions of the VH and VL to human framework sequences, with limited backmutation to rabbit framework sequences. 2 humanized 9C8 scFv weregenerated from the rabbit scFv comparing 2 different VHCDR1 sequences.The rabbit scFv was directly expressed without humanization and thehumanized mAb, and scFvs were expressed by transient transfection ofHEK293 cells. Transfection supernatants were tested for the ability toneutralize 50 pg/ml of human IL-6 using the B9 cell proliferation assay.

FIG. 10 shows a PCR strategy for CDR grafting onto human V-regionframeworks in scFv format. VL, light chain V-region; L, leader (signalpeptide); FR, framework region; CDR, complementarity determining region;arrows indicate individual primers and their directionality in a PCRamplification; CDR-specific primers designated by chain and CDR number,H is VH, L is VL; Primer directionality also designated by F (forward)and R (reverse); curved line between VL-FR4 and VH-FR1 represents the 20aa (G4S)₄ linker that is added for scFv construction.

FIGS. 11A & 11B show IL-6 neutralization by humanized scFv 13A8.Humanized 13A8 anti IL-6 scFv was expressed in mammalian cells. The scFvwas purified from the supernatants by Ni affinity. Testing forneutralization of human IL-6 (B) or primate IL-6 (A) was carried outusing the B9 cell proliferation assay.

FIG. 11C shows IL-6 neutralization by anti IL-6 Humanized scFv:Humanized 9C8 scFv v3-1 (from the multistep method) and 28D2 scFv wereexpressed in mammalian cells, purified by Ni chromatography, andcompared for inhibition of IL-6 induced B9 cell proliferation.

FIG. 12 shows that Anti IL-23 31A12 scFv Neutralizes IL-23 31A12 mAb wasconverted into a humanized scFv and expressed in mammalian transfectionalong with the parental mAb. Both were tested for neutralization of 600pg/ml of eBiosciences human II-23 using the mouse splenocyte assay forinduction of IL-17.

FIG. 13 shows that Humanized Anti IL-23 45G5 scFv Neutralizes HumanIL-23: Humanized 45G5 scFv was compared to the chimeric mAb 31A12 and22H8. All mAbs were expressed in mammalian cells, purified and testedfor the inhibition of 1.2 ng/ml of eBiosciences human IL-23 using themouse splenocyte assay of IL-17 induction.

FIGS. 14A-14D show testing of Humanized 13A8 scFv mammalian expressionconstructs which were engineered to remove the 2 Met residues. Thevarious double mutant constructs, and the parental scFv (MM), wereexpressed in HEK cells and tested for inhibition of 50 pg/ml of humanIL-6 using the in vitro B9 bioassay.

FIG. 14E shows testing of Humanized 31A12 scFv, with both Mets replaced(only H34L versions shown), and the parental Met containing scFv, whichwere expressed transiently in HEK293 cells. Supernatants were tested forinhibition of biological activity of 600 pg/ml of eBiosciences IL-23 inthe mouse splenocyte assay.

FIGS. 14F and 14G show testing of Humanized 45G5 scFv with the H82 Metreplaced with either L or V, both in combination with H34L, which werecompared to the parental 45G5 chimeric mAb and Met free 31A12 scFv(31A12-LL). All were expressed transiently in HEK293 cells. scFv andmAbs were purified and tested for inhibition of biological activity of1200 pg/ml of eBiosciences IL-23 in the mouse splenocyte assay.

FIG. 14H shows that Humanized Anti IL-23 scFvs neutralizes Human IL-23.Wild Type Anti IL-23 scFv 22H8 and 45G5, were compared to the 22H8 scFvwith the Met at H34 replaced with either V or L, as indicated.

FIG. 15 shows neutralization of IL-6 by E. coli 28D2 scFv with Aha atthe N or C term or in the Gly/Ser Linker:

28D2 constructs with a single Met codon at the N or C terminus or in theGly/Ser linker, were expressed in E. coli fermentation, substituting Ahafor Met. These purified scFv were tested for neutralization of 50 pg/mlof human IL-6.

FIG. 16A shows PEGylation of 13A8cAha with 20K linear PEG bis alkyne SDSPAGE (reducing, 4-20% Tris-Glycine) of the 13A8cAha PEGylation reactionwith 20K PEG bis alkyne. Lane 1: 13A8cAha alone; Lane 2: (−) control—noCopper; Lane 3; 200 mL small scale reaction mixture; Lane 4: 600 mLreaction—centrifuged—sample supernatant. Scanning Laser Densitometryindicated a 70% yield (lane 4) of the PEGylated 13A8cAHA.

FIG. 16B shows PEGylation of 31A12cAha with 20K linear PEG bis alkyne.SDS PAGE (reducing, 4-20% Tris-Glycine) of 31A12cAha PEGylation with 20KPEG bis alkyne. Lane 1: Molecular weight markers; Lane 3: (−) control—noCopper, Lane 4: small scale reaction; Lane 5: 400 mLreaction—centrifuged—sample supernatant. Scanning Laser Densitometryindicated a 59% yield (lane 5) of the PEGylated 31A12cAha.

FIG. 16C shows PEGylation of 13A8cAha with 40K linear PEG bis alkyne.SDS-PAGE (reducing, 4-20% Tris-Glycine) of the preparation of 13A8c-40KPEG. Scanning Laser Densitometry indicated a 51% yield

FIG. 16D shows PEGylation of 13A8L Aha with 20K linear PEG Bis-Alkyne.SDS-PAGE (reducing, 4-20% Tris-Glycine). Scanning Laser Densitometryindicated a 60% yield

FIG. 16E shows PEGylation of 45G5cAha with 20K linear PEG bis alkyne.SDS PAGE (reducing, 4-20% Tris-Glycine). Lane 1: (−) control—no Copper;Lane 2: small scale reaction; Lane 3: small scale reaction, no triazoleligand; Lane 4: 160 mL reaction—centrifuged—sample supernatant; Lane 6:Molecular weight markers. Scanning Laser Densitometry indicated a 59%yield of the PEGylated 45G5c.-.

FIG. 17A shows IL-6 Neutralization with 28D2c-PEG. 2 samples of 28D2c-30KPEG refolded under different conditions were assayed for IL-6neutralization.

FIGS. 17B and C shows PEG-scFv Stability: 31A12-PEG has a Tm of 69° C.13A8-PEG has a Tm of 66° C. This is reflected in the stability of thesemolecules in solution as shown. Each scFv-PEG was incubated in PBS (orTween as indicated) and is assayed for potency relative to the parentalscFv (from mammalian expression). Storage temperature, for 13 or 20days, is indicated; 3×FT indicates three cycles of freezing and thawing.

FIG. 18A shows the preparation of 13A8c-PEG-31A12c Bispecific SDS-PAGE(reducing, 4-20% Tris-Glycine). Lane 1: Molecular weight markers, Lane2: 13A8-PEG alone, Lane 3: (−) control—no copper, Lane4: 1000 mLreaction

FIG. 18B shows the preparation of 13A8n-PEG-45G5c Bispecific: SDS-PAGE(reducing, 4-20% Tris-Glycine). Lane 1: Large Scale Reaction 1, Lane 3:Large Scale Reaction 2

FIG. 18C shows the preparation of 13A8c-PEG-22H8c Bispecific SDS-PAGEreducing, 4-20% Tris-Glycine). Lane 1: Molecular weight markers, Lane 2:13A8c-PEG alone, Lane 3: (−) control—no copper, Lane4: 1150 mL reaction

FIG. 18D shows the preparation of 13A8c-40 KPEG-31A12cAHA. SDS-PAGE(reducing 4-20% Tris-Glycine). Lane 1: MW markers, Lane 2: 13A8c-40KPEG, Lane 3: direct sample of reaction mixture, Lane 4: sample of finalprocessed mixture 6 uL load Lane 5: sample of final processed mixture 12uL load. Lane 6: Reaction with no copper, Lane 87: 31A12cAHA alone. Theyield (average of 2 loads)=56% with a product to monovalent ratio of4.5:1.

FIG. 18E shows the preparation of 13A8L-PEG-31A12c Bispecific SDS-PAGE(reducing, 4-20% Tris-Glycine). Lane 1 reaction of 31A12-20KPEG+13A8LAha to form bispecific. Reaction yield was found to be 37%.

FIG. 19A shows the functional Activity of 31A12c-PEG-13A8c forNeutralization of IL-6 and IL-23 The bioactivity of bispecific vs IL 6and IL23 with comparison to scFv alone. A: Anti-IL-6 activity for 13A8scFv portion of the bispecific. 19B: Anti-IL-23 activity for the 31A12cscFv portion of the bispecific was also measured. The EC50s werecalculated from the titrations.

FIG. 20A shows Rat PK of 28D2c scFv administered SC. Rats were treatedSC with 1 mg/kg of anti IL-6 scFv 28D2c. Blood was collected at theindicated times, the presence of 28D2 in the plasma of the rats wasmeasured using an anti IL-6 neutralization assay.

FIG. 20B shows Rat PK of 31A12c-PEG-13A8c bispecific administeredsubcutaneously: Rats were treated SC with 1 mg/kg of 31A12c-PEG-13A8cbispecific. Blood was collected at the indicated times, the presence ofbispecific in the plasma of the rats was measured using an anti IL-6neutralization assay. The PK data for the 28D2 scFv from FIG. 20B isincluded here for comparison.

FIG. 21 shows the bioactivity of the 13A8n-PEG-31A12c bispecific. Theneutralization of 50 pg/ml of IL-6 by the bispecific was measured in theB9 bioassay. The mammalian 13A8 scFv protein is included for comparison.

FIG. 22 shows the functional activity of 13A8n-PEG-45G5 for IL-6 andIL-23. The bioactivity of bispecific vs IL 6 and IL23 is shown. Theneutralization of 50 pg/ml of human IL-6 (22A) and 1200 pg/ml (22B) ofhuman eBiosciences IL-23, by the 13A8n-PEG-45G5 bispecific was measuredusing the B9 cell line bioassay for IL-6 and the mouse splenocyte assayfor IL-23. EC50 values were calculated from the curves.

FIG. 23 shows the activity of 13A8c-PEG-22H8c for IL-6 and IL-23.Bioactivity of 13A8c-PEG-22H8c for IL-6 and IL-23 is shown. Theneutralization of 50 pg/ml of human IL-6 (A) and 1200 pg/ml (B) of humaneBiosciences IL-23, by the 13A8c-PEG-22H8 bispecific was measured usingthe B9 cell line bioassay for IL-6 and the mouse splenocyte assay forIL-23. EC50 values were calculated from the curves.

FIG. 24 shows the activity of 13A8c-40 KPEG-31A12c for IL-6 and IL-23.Bioactivity of 13A8c-40kPEG31A12c for IL-6 and IL-23 is shown. Theneutralization of 50 pg/ml of human IL-6 (A) and 1200 pg/ml (B) of humaneBiosciences IL-23, by the bispecific was measured using the B9 cellline bioassay for IL-6 and the mouse splenocyte assay for IL-23. EC50values were calculated from the curves.

FIG. 25 A Shows serum levels (as measured in the B9 assay) of the13A8c-40 KPEG-31A12c bispecific, 13A8c-20 KPEG-31A12c bispecific,13A8c-PEG and a naked scFv (28D2) after subcutaneous administration inrats.

FIG. 25 B Shows the results of pharmacokinetic analysis of serum levelsof 13A8c-40 KPEG-31A12c bispecific, 13A8c-20 KPEG-31A12c bispecific,13A8c-PEG and naked scFv (28D2) after subcutaneous administration inrats.

FIG. 26A shows in vitro polarization of T_(H)17/22 cells Different humanT cell subsets, including T_(H)17 and T_(H)22 cells, can be generated inboth in vivo and in vitro systems.

FIG. 26B shows in vitro Human Th17 Development: Human PBMC werestimulated with anti-CD3/28 for 7 days in vitro either alone or in thepresence of LPS and TGFb, as indicated. They were then restimulated withPMA+ ionomycin as indicated and stained for IL-17 and RORC FIG. 26Cshows T_(H)17 and T_(H)22Cells can be generated from cultured PBMCT_(H)17 are seen in the mixed lymphocyte reaction, while T_(H)17 andT_(H)22 are seen with anti CD3 stimulated PBMC. PBMC were stimulated for5 d with anti CD3/28+IL-1b+LPS or allogeneic PBMC+ peptidoglycan, thenrestimulated with PMA+ lonomycin and stained for intracellular for IL-17and IL-22.

FIG. 27 shows inhibition of Th17 and Th22 development in vitro withselected scFvs. Human PBMC were cultured for 5 days in anti CD3+ antiCD28 and LPS+IL-1+TGFb in the presence of the indicated scFv. After 5days, the cells were restimulated with PMA+ ionomycin and the % of CD4,IL-17 and IL-22 producing cells was determined by flow cytometry.

FIG. 28 shows a mixed lymphocyte reaction. Inhibitory effect of antiIL-6 and IL-23 scFv used alone or used in combination, on T_(H)17differentiation is shown. The indicated anti IL-6 scFv and anti IL-23scFv were added to PBMC cultures during stimulation with allogeneicPBMC. After 5 days, the cells were washed and restimulated with PMA+lonomycin and stained for IL-17.

FIG. 29 shows the beneficial inhibitory effect of bispecific antiIL-6/IL-23 antibodies on T_(H)17 differentiation. Anti IL-6 and antiIL-23 mAbs (13A8 and 31A12) were tested alone or in combination, as wellas 31A12c-20 KPEG-13A8c bispecific. The mAbs or the bispecific wereadded to PBMC cultures during stimulation with allogeneic PBMC. Molarconcentration of binding domains added is indicated. After 5 days, thecells were washed and restimulated with PMA+ lonomycin and stained forIL-17.

FIG. 30 shows in-vivo polarization of T_(H)17/22 cells Different human Tcell subsets, including T_(H)17 and T_(H)22 cells, can be generated inboth in vivo and in vitro systems.

FIG. 31A shows treatment of humanized scid/hu mice with a combination ofantagonists against IL-6 and IL-23. NSG mice that were successfullyengrafted with human immune cells, were transplanted with humanallogeneic skin and received 100 mg of 13A8c-PEG anti IL-6 and31A12c-PEG anti IL-23 (scFv-PEGs) every 2 days. Thirty days after skintransplant, spleens were recovered and single cell suspensions werestimulated with PMA/ionomycin and assayed for intracellular cytokines.CD3+/CD4+ cells were analyzed for IL-17 and IL-22 production by flowcytometry

FIG. 31B shows intracellular cytokine expression in CD3+/CD4+ cells fromspleens of humanized scid/hu mice treated with a combination ofantagonists against IL-6 and IL-23. As described in the previous Figurethe splenocytes form treated and untreated NSG mice with skinallografts, CD3+/CD4+ cells were analyzed for intracellular IL-17 andIL-22 by flow cytometry. Data shows marked reductions in all populationsof IL-17 and IL-22 positive CD4+ T cells in animals treated with antiIL-6 and anti IL-23. The data are plotted as the mean and SEM of thetreated or untreated mice according to the indicated subset of TH17 orTH22 cells.

FIG. 32 shows the effect of the 13A8cPEG-31A12c bispecific on inhibitionof Th17 and Th22 differentiation in Sci/hu allograft model:

Adult scid mice with established human immune systems were transplantedwith allogeneic human skin. After 4 weeks of EOD treatment, asindicated, the splenocytes were activated in vitro and the cytokinesmeasured in each human CD4 T cell by multi-parameter flow cytometry.Each point indicates an individual treated or control mouse, and eachmouse is represented 3 times (for each cytokine shown).Monospecific scFv anti-IL23 is 31A12cPEG; bispecific scFv anti IL6 IL23is 13A8c-20 KPEG-31A12c. Untreated mice received placebo. 13A8c-20KPEG-31A12c significantly reduced the differentiation of Th17 cells asmeasured by the inhibition of IL-17 (FIG. 32A, *p<0.05) and IL-22 (FIG.32B, p<0.05) producing CD4⁺ human T cells. All other panels measuregeneral leukocyte markers and indicate that 13A8c-20 KPEG-31A12c is notgenerally immunosuppressive to leukocytes other than TH17/22 cells.

FIG. 33 shows histological analysis of a section of epidermis of placebotreated mice (A) compared to mice treated with 13A8c-20kPEG-31A12c antiIL-6/anti IL-23 bispecific (B), in which the 13A8c-20kPEG-31A12c antiIL-6/anti IL-23 bispecific significantly reduces the histologicalfeatures of psoriasis, epidermal thickness in particular.

FIG. 34 shows Six experiments utilizing the scid/hu allograft model werecompleted and the clinical scores judged by a pathologist blinded duringthe treatment period and are summarized in FIG. 34A (clinical scores).The analysis of histological sections enables a highly quantitativemeasurement of the most meaningful metrics of psoriasis, in particular,epidermal thickness which is an unbiased measure is shown if FIG. 34B(quantitative epidermal thickness). The bispecific scFv has a highlysignificant and potent effect on the reduction of psoriasis clinicalscores and epidermal thickness.

FIG. 35 A shows the readout of the ear hyperplasia mouse model: Micereceived intra-dermal injections of rhIL-23 in the right ear (1 μg) in avolume of 20 μL on days 0, 1, 2 and 3. PBS was injected intocontra-lateral ear as control. Ear thickness was measured on day 4. Thefirst panel shows ear thickness of the IL-23 injected ear compared tothe PBS injected ear. The second panel shows the increase in thicknessof the IL-23 injected ear compared to the PBS injected ear for eachanimal.

FIG. 35 B shows the results of the ear hyperplasia model when mice weretreated with vehicle or 13A8c-20 KPEG-31A12c (100 ug i.p.) on days −1and 2. The first panel shows ear thickness of IL-23 injected earscompared to PBS injected ears in both the vehicle or 13A8c-20KPEG-31A12c treated animals. The second panel shows the increase in earthickness when comparing the IL-23 injected ear to the PBS injected earfor each animal.

FIG. 35 C shows the results of the ear hyperplasia model when mice weretreated with vehicle or 13A8c-20 KPEG-31A12c or 13A8c-40 KPEG-31A12c(100 ug i.p.) on day −1 only. The first panel shows ear thickness ofIL-23 injected ears compared to PBS injected ears in both the vehicle,13A8c-20 KPEG-31A12c or 13A8c-40 KPEG-31A12c treated animals. The secondpanel shows the increase in ear thickness when comparing the IL-23injected ear to the PBS injected ear for each animal.

FIG. 35 D shows the results of the ear hyperplasia model when mice weretreated with vehicle or 13A8c-40 KPEG-31A12c (100 ug i.p.) orUstekinumab (288 ug i.p.) on days −1 and 2. The first panel shows earthickness of IL-23 injected ears compared to PBS injected ears in boththe vehicle, 13A8c-40 KPEG-31A12c or Ustekinumab treated animals. Thesecond panel shows the increase in ear thickness when comparing theIL-23 injected ear to the PBS injected ear for each animal.

FIG. 36 A shows binding of anti IL-23 chimeric antibodies to IL-12coated on ELISA plates. An anti IL-6 antibody (13A8) is included as anegative control. In the first panel human IL-12 is coated on the plate.22H8 shows strong binding while 31A12 and 49B7 show weaker, but stillpositive binding. In the second panel monkey IL-12 (macaque) is coatedon the plates. Here 49B7, 31A12 and 22H8 all show strong binding tomacaque IL-12. In the third panel the plates are coated with human IL-12p40 subunit. 22H8 shows strong binding, and 49B7 and 31A12 weakerbinding to the p40 subunit.

FIG. 36 B shows neutralization of macaque IL-12 induced Interferonγsecretion in the NK92 cell bioassay. Both 31A12 and 22H8 show stronginhibition of the macaque IL-12.

FIG. 36 C shows the neutralization of human IL-12 induced Interferonγsecretion in the NK92 cell bioassay. Here, in contrast to the macaqueIL-12, human IL-12 is not neutralized by 31A12 or 49B7. 22H8 neutralizesboth macaque and human IL-12.

DETAILED DESCRIPTION OF THE INVENTION Bispecific Constructs

The specification describes, inter alia, bivalent, bispecific constructsthat bind to IL-6 and IL-23 and modulate their activity. IL-6 and IL-23are both known to play a role in the differentiation and activation ofT_(H)17 cells. The activated T_(H)17 cells are in turn involved inmediating immune responses through a variety of downstream pathways.These two cytokines function at different stages of T_(H)17differentiation with IL-6 acting very early in T cell commitment of theT_(H)17 pathway and IL-23 acting on committed T_(H)17 cells. Thus, thepresent invention provides novel bivalent bispecific constructs thatinhibit two distinct points in the T_(H)17 activation pathway and haveadditional inhibitory effects on some of the downstream inflammatoryresponses mediated by T_(H)17 products (e.g. in fibroblasts, endothelialcells, epithelial cells and stromal cells). By targeting both IL-6 andIL-23, the bispecific molecules are able to inhibit T_(H)17 mediatedresponses at multiple points in the T_(H)17 pathway and potentially actwith greater potency than the corresponding monospecific antibodiesalone. The person skilled in the art will appreciate that the successfulproduction of stable bivalent, bispecific construct that retains thefunctional characteristics of its constituent antibodies, or hasimproved functional characteristics, represents a surprising andunexpected result given the uncertainties involved in generatingbivalent, bispecific antibodies.

In addition, the bivalent bispecific constructs of the present inventioncan modulate (e.g. inhibit), T_(H)22 cell activation. T_(H)22 representa recently identified (Eyerich et al, 2009), distinct subset of T helpercells that are involved in inflammatory and wound healing processes andare particularly implicated in skin inflammation (Nograles et al, 2009).The mechanism of their activation and subsequent action remains thesubject of investigation, but the cells themselves are characterized bythe secretion of IL-22 and TNF-α but not IL-17 or Interferonγ. Th22cells have not been fully characterized, but can be isolated frompatients with psoriasis, and express a distinctive gene expressionprofile from that seen with other T cell subsets. IL-22 expression hasbeen reported to be IL-23 dependent (Kreymborg et al, 2007). The studiesconducted here further suggest that Th22 cells are IL-2 dependent incontrast to Th17 cells which rely on IL-21 for growth stimulation.

The antibodies and bivalent bispecific constructs of the presentinvention may be specific for either IL-23 or for both IL-23 and IL-12.Thus, the present invention provides a subset of antibodies and bivalentbispecific constructs that bind IL-23, which also target IL-12molecules. Without wishing to be bound by theory it is likely that thissubset of anti-IL-23 antibodies (referred to herein as anti-IL-23/IL-12antibodies) may bind the p40 subunit common to both IL-12 and II-23 (seee.g. FIG. 10). Those that target the p40 subunit of IL-23 are likely toinhibit IL-12 in addition to IL-23. Furthermore, antibodies against p40may bind an epitope which impairs IL-23 activity without inhibitingIL-12 activity. In contrast, those antibodies that target the p19subunit of IL-23 would not be expected to bind IL-12. IL-12 is involvedin T_(H)1 mediated immune responses and as such these particularbivalent bispecific constructs may be useful in modulating not onlyT_(H)17 cell mediated immune responses but also T_(H)1 cell mediatedresponses. This may be particularly advantageous in treating conditionsthat have both a T_(H)1 mediated and T_(H)17 mediated aspect to theiraetiology.

Thus, in an embodiment the bivalent, bispecific constructs of thepresent invention comprise an anti-IL-6 antibody, or derivative thereof,and an anti-IL-23 antibody, or derivative thereof.

In another embodiment the bivalent, bispecific constructs of the presentinvention comprise an anti-IL-6 antibody, or derivative thereof, and ananti-IL-23/IL-12 antibody, or derivative thereof.

Particular examples of the bivalent, bispecific constructs of thepresent invention can be assayed for their utility in modulating bothIL-23 and IL-6 activity using both in vitro and in vivo methods. Inparticular, the assays detailed below may be used.

The components of the bivalent bispecific constructs and their means ofidentification and manufacture are discussed further below.

Generation of Parent Anti-IL-6, Anti-IL-23 and Anti-IL-23/IL-12Antibodies

The initial antibodies on which the antibodies, and derivatives thereof,in the bivalent bispecific constructs of the present invention are basedcan be identified by standard experimental techniques. These antibodiesare referred to herein as parent antibodies.

Selection of Parent Antibodies

In an embodiment the parent antibodies are selected on the basis oftheir ability to bind IL-6, IL-23 or IL-12. The binding of the parentantibodies can be measured by determining their Kd values. In anotherembodiment the parent antibodies are selected on the basis of theirability to modulate the activity of IL-6, IL-23 or IL-12. In a preferredembodiment the parent antibodies are selected on the basis of theirability to bind IL-6, IL-23 or IL-12, and on their ability to modulatethe activity of IL-6, IL-23 or IL-12. The parent antibodies may beselected for their ability to inhibit the biological activity of IL-6,IL-23 or II-12, or they may be selected for their ability to promote thebiological activity of IL-6, IL-23 or IL-12. Preferably, the parentantibodies are selected for their ability to inhibit IL-6, IL-23 andIL-12.

Sources of Parent Antibody

In an embodiment, the parent antibodies, or derivatives thereof, can beobtained from identical or separate animal species.

The parent antibodies may, for example, be obtained from an antibodyproduced in primate, rodent, lagomorph, tylopoda or cartilaginous fish.

The parent antibodies may be obtained from transgenic animals. Forinstance, they may be obtained from a transgenic mouse that has beengenetically altered to possess a human immune system, e.g. a Xenomouse®.Antibodies produced in such transgenic animals may have thecharacteristics of antibodies produced by the exogenous immune system,e.g. antibodies from a Xenomouse may be regarded as human antibodies.

In the event that one or more of the parent antibodies are obtained froma rodent, the rodent is advantageously a, mouse or a rat.

In the event the antibody is obtained from a lagomorph, the lagomorph isadvantageously a rabbit.

In the event that one or more of the parent antibodies are obtained froma tylopoda they be obtained from a camel, a llama or a dromedary. Thisuse of such “camelid” antibodies may be advantageous as these speciesare known to produce high affinity antibodies of only a single variabledomain. In the event that a tylopoda antibody is used, it isadvantageous to use the VHH domain or a modified variant thereof.

In the event that one or more of the parent antibodies are obtained froma cartilaginous fish, the cartilaginous fish is advantageously a shark.

In the event that one or more of the parent antibodies are obtained froma primate, the primate is advantageously a monkey or ape.

Immortalisation of Antibodies

The parent antibodies may be immortalized by standard experimentaltechniques. As such the present invention provides monoclonal antibodiesgenerated from the parent antibodies that are suitable for incorporationinto a bivalent bispecific construct according to the present invention.

Combinations of Particular Antibodies

The present invention also provides compositions comprising acombination of the antibodies and/or derivatives thereof. Thecombinations comprise an IL-6 antibody, or derivative thereof, and anIL-23 antibody or derivative thereof. The IL-23 antibody, or derivativethereof, may also bind IL-12.

Preferred combinations of antibodies, and derivatives thereof, compriseany one of the IL-6 antibodies defined below, combined with any one ofthe anti-IL-23 or anti-IL-23/IL-12antibodies defined below.

The compositions comprising such combinations are expected to havegreater activity than the individual antibodies when administered alone.A particularly preferred combination of antibodies, or derivativesthereof, is the anti-IL-6 antibody, 13A8, or a derivative based on 13A8and the anti-IL-23 antibody, 31A12, or a derivative based on 31A12. Thiscombination of antibodies provides greater inhibition of T_(H)17 cellactivity compared to either antigen alone. The combination has greaterT_(H)17 cell inhibitory activity than antibodies known in the art.Furthermore, it exhibits this inhibitory activity at advantageously lowdosages.

A particularly preferred combination of antibodies or derivativesthereof, comprises a PEGylated IL-6 antibody or derivative thereofcombined with a PEGylated IL-23 antibody or derivative thereof. TheIL-23 antibody, or derivative thereof, may also bind IL-12 (i.e. be anIL-23/IL-12 antibody).

Humanization of Antibodies

The antibodies of the bivalent bispecific construct may be subjected toalteration to render them less immunogenic when administered to a human.Such an alteration may comprise one or more of the techniques commonlyknown as chimerization, humanization, CDR-grafting, deimmunizationand/or mutation of framework region amino acids to correspond to theclosest human germline sequence (germlining). Subjecting antibodies tosuch alteration has the advantage that an antibody which would otherwiseelicit a host immune response is rendered more, or completely“invisible” to the host immune system, so that such an immune responsedoes not occur or is reduced. Antibodies which have been altered asdescribed according to this embodiment will therefore remainadministrable for a longer period of time with reduced or no immuneresponse-related side effects than corresponding antibodies which havenot undergone any such alteration(s). One of ordinary skill in the artwill understand how to determine whether, and to what degree an antibodymust be altered in order to prevent it from eliciting an unwanted hostimmune response.

Thus the present invention provides humanized, or chimeric antibodiesthat have been altered such that they include amino acid sequences fromone or more organisms, or contain synthetic amino acid sequences (e.g. ahumanized or chimeric antibody according to the present invention maycomprise human framework regions joined to CDR regions obtained from arodent).

Particular Antibodies of Interest

Thus according to the present invention particular humanized anti-IL-6,anti-IL-23 and anti-IL-23/IL-12 antibodies are provided. Theseantibodies are based on parent antibodies that demonstrated the abilityto both bind IL-6, IL-23 or p40 and to modulate (e.g. inhibit) theirbiological activity. Furthermore, the particular antibodies provided bythe present invention retain, or substantially retain, these abilitiesfollowing immortalization and humanization.

Particular humanized antibodies of interest include the following:

Anti-IL-6 Antibodies:

13A8 (comprising the VH of SEQ ID NO. 259 and the VL of SEQ ID NO. 261);9H4 (comprising the VH of SEQ ID NO. 46 and the VL of SEQ ID NO. 48)9C8 (comprising the VH of SEQ ID NO. 56 and the VL of SEQ ID NO. 58)8C8 (comprising the VH of SEQ ID NO. 36 and the VL of SEQ ID NO. 38)18D4 (comprising the VH of SEQ ID NO. 26 and the VL of SEQ ID NO. 28);and28D2 ((comprising the VH of SEQ ID NO. 16 and the VL of SEQ ID NO. 18).

Anti-IL-23 Antibodies:

31A12 (comprising the VH of SEQ ID NO. 267 and the VL of SEQ ID NO.269);34E11 (comprising the VH of SEQ ID NO. 116 and the VL of SEQ ID NO.118);35H4 (comprising the VH of SEQ ID NO. 126 and the VL of SEQ ID NO. 128);49B7 (comprising the VH of SEQ ID NO. 96 and the VL of SEQ ID NO. 98);and16C6 (comprising the VH of SEQ ID NO. 106 and the VL of SEQ ID NO. 108).

Anti-IL-23/IL-12 Antibodies:

45G5 (comprising the VH of SEQ ID NO. 275 and the VL of SEQ ID NO. 277);14B5 (comprising the VH of SEQ ID NO. 186 and the VL of SEQ ID NO. 188)4F3 (comprising the VH of SEQ ID NO. 166 and the VL of SEQ ID NO. 168)5C5 (comprising the VH of SEQ ID NO. 176 and the VL of SEQ ID NO. 178)22H8 (comprising the VH of SEQ ID NO. 271 and the VL of SEQ ID NO. 273);and1H1 (comprising the VH of SEQ ID NO. 156 and the VL of SEQ ID NO. 158)Particularly preferred humanized antibodies are humanized forms of 13A8,31A12 and 22H8.

Antibody Variants

The present invention also provides antibody variants, for example, ascomponents of the bivalent, bispecific construct. The antibodies retain,or substantially retain, the binding affinity and ability to modulatethe biological activity of IL-6, IL-23 or IL-12 (e.g. the Kd value of avariant antibody is at least 80% compared to its parent antibody, andits ability to modulate biological activity is at least 80% of that ofits parent antibody as determined by the assays disclosed herein).

Variant antibodies or derivatives thereof may be obtained by mutatingthe variable domains of the heavy and/or light chains to alter a bindingproperty of the antibody. For example, a mutation may be made in thenucleic acid molecule encoding one or more of the CDR regions toincrease or decrease the Kd of the antibody for IL-6 or IL-23, toincrease or decrease the ability of the antibody to modulate thebiological activity of IL-6, IL-23 or IL-12, or to alter the bindingspecificity of the antibody. Techniques for introducing such mutationsusing site-directed mutagenesis are well-known in the art.

Further variant antibodies or derivatives thereof may be obtained bymutating the variable domains of the heavy and/or light chains to alterthe isoelectric point (p1) to enhance protein stability at the pH 3-7.5range of the final formulation to avoid disulphide bond shuffling. Seefor example SEQ ID NO 332, 31A12 pl optimization where the followingaminoacids were modified: Q26R, L56R, K109-G110insR, and Q142K; SEQ ID334, 13A18 pl optimization where the following aminoacids were modified:Q26R, L56R, K112-G113insR, and Q145K.

Furthermore, stability may be enhanced by mutating the variable domainsof the heavy and/or light chains to reduce aggregation of the product insolution, see for example SEQ ID NO 331, 31A12 F125 mutation predictedto enhance solubility and reduce aggregation of the product, and SEQ IDNO. 333, 31A12 combined pl optimization and F125 mutation

In another embodiment, the nucleic acid molecules may be mutated in oneor more of the framework regions. A mutation may be made in a frameworkregion or constant domain to increase the half-life of the anti-IL-6 oranti-IL-23 antibody. A mutation in a framework region or constant domainmay also be made to alter the immunogenicity of the antibody, to providea site for covalent or non-covalent binding to another molecule, or toalter such properties as complement fixation.

Thus, according to the present invention mutations may be made in eachof the framework regions, the constant domain and the variable regionsin a single mutated antibody. Alternatively, mutations may be made inonly one of the framework regions, the variable regions or the constantdomain in a single mutated antibody.

Sequence Variation

In an embodiment, the present invention provides variant anti-IL-6antibodies that have at least 90% sequence identity to the anti-IL-6antibody prior to mutation. Preferably the variant anti-IL-6 antibodyhas at least 95%, 96%, 97%, 98% or 99% sequence identity to theanti-IL-6 antibody prior to mutation.

In an embodiment, the present invention provides variant anti-IL23antibodies that have at least 90% sequence identity to the anti-IL-23antibody prior to mutation. Preferably the variant anti-IL-23 antibodyhas at least 95%, 96%, 97%, 98% or 99% sequence identity to theanti-IL23 antibody prior to mutation.

In an embodiment, the present invention provides variantanti-IL-23/IL-12 antibodies that have at least 90% sequence identity tothe anti-IL-23/IL-12 antibody prior to mutation. Preferably the variantanti-IL-23/IL-12 antibody has at least 95%, 96%, 97%, 98% or 99%sequence identity to the anti-IL-23/IL-12 antibody prior to mutation.

Addition Deletion Substitution

In one embodiment, there are no greater than ten amino acid changes ineither the VH or VL regions of the variant anti-IL-6 antibody comparedto the anti-IL-6 antibody prior to mutation.

In another embodiment, there are no greater than ten amino acid changesin either the VH or VL regions of the variant anti-IL-23 antibodycompared to the anti-IL-23 antibody prior to mutation.

In another embodiment, there are no greater than ten amino acid changesin either the VH or

VL regions of the variant anti-IL-23/IL-12 antibody compared to theanti-IL-23/IL-12 antibody prior to mutation.

In a more preferred embodiment, there are no more than five amino acidchanges in either the VH or VL regions of the variant anti-IL-6antibody, in the variant anti-IL-23 antibody or in the variantanti-IL-23/IL-12 antibody, more preferably no more than three amino acidchanges. In another embodiment, there are no more than fifteen aminoacid changes in the constant domains of either the variant anti-IL-6antibody compared to the anti-IL-6 antibody prior to mutation, thevariant anti-IL-23 antibody compared to the anti-IL-23 antibody prior tomutation, or the variant anti-IL-23/IL-12 antibody compared to theanti-IL-23/IL-12 antibody prior to mutation, more preferably, there areno more than ten amino acid changes, even more preferably, no more thanfive amino acid changes.

Antibody Derivatives

Antibody derivatives may be generated using techniques and methods knownto one of ordinary skill in the art. Antibody derivatives according tothe present invention retain, or substantially retain, the bindingaffinity and ability to modulate the biological activity of IL-6, IL-23or p40 of the antibodies from which they are derived. Examples ofantibody derivatives include, Fab, Fab′, F(ab)′ and scFv constructs,Kappabodies, Minibodies, and Janusins derived from the anti-IL-6,anti-IL-23, and anti-IL-23/IL-12 antibodies disclosed herein.

Fab, Fab′ F(ab)′

In an embodiment of the present invention Fab, Fab′, F(ab)′ fragments ofthe anti-IL-6 antibodies or variant anti-IL-6 antibodies are provided.

In an embodiment of the present invention Fab, Fab′, F(ab)′ fragments ofthe anti-IL-23 antibodies or variant anti-IL-23 antibodies are provided.

In an embodiment of the present invention Fab, Fab′, F(ab)′ fragments ofthe anti-IL-23/IL-12-23/IL-126 antibodies or variant anti-IL-23/IL-12antibodies are provided.

Single Chain Antibodies (scFv)

In an embodiment of the present invention scFv derivatives of theanti-IL-6 antibodies or variant anti-IL-6 antibodies are provided.

In an embodiment of the present invention scFv derivatives of theanti-IL-23 antibodies or variant anti-IL-23 antibodies are provided.

In an embodiment of the present invention scFv derivatives of theanti-IL-23/IL-12 antibodies or variant anti-IL-23/IL-12 antibodies areprovided.

To create a single chain antibody (scFv), the VH- and VL-encoding DNAfragments are operatively linked to another fragment encoding a flexiblelinker such that the VH and VL sequences can be expressed as acontiguous single-chain protein, with the VL and VH regions joined bythe flexible linker (see e.g., Bird et al. (1988) Science 242: 423-426;Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883;McCafferty et al., Nature (1990) 348: 552-554). The single chainantibody may be monovalent, if only a single VH and VL are used,bivalent, if two VH and VL are used, or polyvalent, if more than two VHand VL are used.

In an embodiment, the single chain antibody is prepared using one ormore of the variable regions from an anti-IL-6 antibody. In anotherembodiment, the single chain antibody is prepared using one or more CDRregions from the anti-IL-6 antibody.

In one embodiment, the single chain antibody is prepared using one ormore of the variable regions from an anti-IL-23 antibody. In anotherembodiment, the single chain antibody is prepared using one or more CDRregions from the anti-IL-23 antibody.

In one embodiment, the single chain antibody is prepared using one ormore of the variable regions from an anti-IL-23/IL-12 antibody. Inanother embodiment, the single chain antibody is prepared using one ormore CDR regions from the anti-IL-23/IL-12 antibody.

In a preferred embodiment anti-IL-6 single chain antibodies are derivedfrom the humanized anti-IL-6 antibodies described above

In a preferred embodiment anti-IL-23 single chain antibodies are derivedfrom the humanized anti-IL-23 antibodies described above

In a preferred embodiment anti-IL-23/IL-12 single chain antibodies arederived from the humanized anti-IL-23/IL-12 antibodies described above.

In an embodiment the light and heavy chains of the single chainantibodies are joined by a linker portion having the following aminoacid sequences

(SEQ ID NO. 327) GGGGSGGGGSGGGGSGGGGS, (SEQ ID NO. 328) GGGGSGGGGSGGGGS,(SEQ ID NO. 329) GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO. 330)GGGGSGGSGGGGSGGGGS

A linker portion of the present invention may be the sequence GGGGSrepeated 3 to 5 times, or a non integer repeat of the GGGGS sequence,see for instance SEQ ID NO. 330.

In an embodiment the single chain antibodies of the invention arecovalently linked to PEG.

Kappabodies, Minibodies, and Janusins

In another embodiment, other modified antibodies may be prepared usinganti-IL-6 antibody, anti-IL-23-antibody or anti-IL-23/IL-12 antibodyencoding nucleic acid molecules. For instance, “Kappa bodies” (ILI etal., Protein Eng 10: 949-57 (1997)), “Minibodies” (Martin et al., EMBO J13: 5303-9 (1994)), or “Janusins” (Traunecker et al., EMBO J 10:3655-3659 (1991) and Traunecker et al. “Janusin: new molecular designfor bispecific reagents” Int J Cancer Suppl 7: 51-52 (1992)) may beprepared using standard molecular biological techniques.

Complementarity Determining Regions (CDRs)

Complementarity determining regions (CDRs) are relatively short aminoacid sequence in the shape of a flexible loop, found in the variable (V)domains of antigen receptors (e.g. immunoglobulin and T cell receptor).The CDRs of both immunoglobulin and the T cell receptor are the parts ofthese molecules that determine their specificity and make contact with aspecific ligand. The CDRs are the most variable part of the molecule,and contribute to the diversity of these molecules, allowing theimmunoglobulin and the T cell receptor to recognize a vast repertoire ofantigens. As such these regions in the anti-IL-6, anti-IL-23 andanti-IL-23/IL-12 antibodies that make up the bivalent, bispecificconstructs of the invention play a key role in determining thespecificity of the antibodies, and antibodies that have particular CDRsregions in common would be expected to have the same or similar antigenspecificity. Thus in an aspect of the invention the anti-IL-6,anti-IL-23 and anti-IL-23/IL-12 antibodies, or derivatives thereof,comprise the CDR regions of the antibodies on which they are based.

It should also be noted that some CDR regions are believed to play amore critical role in antibody specificity than others. In particular,it is often advantageous to use at least the third complementaritydetermining region (CDR) of the VH domain, as these are known to play amajor role in the specificity and affinity of binding of all the CDRregions, in designing an antibody or derivative thereof for inclusion ina bivalent bispecific construct. Thus the present invention provides forthe antibodies and antibody derivatives that make up the bivalent,bispecific constructs of the invention to comprise at least one of CDR1,CDR2, CDR3, CDR4, CDR5 and CDR6 of a parent antibody. Preferably, theantibodies and antibody derivatives that that make up the bivalent,bispecific constructs comprise at least CDR3.

In an embodiment the mutated anti-IL-6 antibody has at least onecomplementarity determining region (CDR) that remains unchanged comparedto the anti-IL-6 antibody prior to mutation. The unchanged CDR may beCDR1, CDR2, CDR3, CDR4, CDR5 or CDR6.

In another embodiment the mutated anti-IL-23 antibody has at least onecomplementarity determining region (CDR) that remains unchanged comparedto the anti-IL-23 antibody prior to mutation. The unchanged CDR may beCDR1, CDR2, CDR3, CDR4, CDR5 or CDR6.

In an embodiment the mutated anti-IL-23/IL-12 antibody has at least onecomplementarity determining region (CDR) that remains unchanged comparedto the anti-IL-6 antibody prior to mutation. The unchanged CDR may beCDR1, CDR2, CDR3, CDR4, CDR5 or CDR6.

Motifs within Amino Acid Sequences of the CDRs

It will be appreciated by the person skilled in the art that even withindividual CDRs there are particular regions (referred to herein asmotifs) that are particularly important in determining the specificityof a particular antibody or derivative thereof. The specificity of theseregions may be determined by a number of factors, such as theirconformation and the location of charged amino acid residues within theregion. The person skilled in the art may identify these motifs throughtechniques known in the art including, epitope mapping and comparing thesequence of antibodies known to bind the same target. Thus the presentinvention provides antibodies or derivatives thereof that comprise CDRshaving particular motifs.

In an embodiment the CDRs comprise at least 3, at least 4, at least 5 orat least 6 consecutive amino acids taken from the CDR regions of thefollowing antibodies:

13A8 (CDRs of SEQ ID NO. 10-15); 9H4 (CDRs of SEQ ID NO. 50-55); 9C8(CDRs of SEQ ID NO. 60-65); 8C8 (CDRs of SEQ ID NO. 40-45); 18D4 (CDRsof SEQ ID NO. 30-35); 28D2 (CDRs of SEQ ID NO. 20-25); 31A12 (CDRs ofSEQ ID NO. 90-95); 34E11 (CDRs of SEQ ID NO. 120-125); 35H4 (CDRs of SEQID NO. 130-135); 49B7 (CDRs of SEQ ID NO. 100-105); 16C6 (CDRs of SEQ IDNO. 110-115); 45G5 (CDRs of SEQ ID NO. 150-155); 14B5 (CDRs of SEQ IDNO. 190-195); 4F3 (CDRs of SEQ ID NO. 170-175); 5C5 (CDRs of SEQ ID NO.180-185); 22H8 (CDRs of SEQ ID NO. 140-145); and 1H1 (CDRs of SEQ ID NO.16-165).

In another embodiment the CDRs comprise substituted consecutive aminoacid sequences taken from the above mentioned CDRs. In particular, themotifs may comprise at least 3, at least 4, at least 5, or at least 6residues wherein the identity and position of the amino acid is fixedrelative to the other amino acids in the sequence, and one or two aminoacids may be substituted, compared to the corresponding amino acid ofthe CDR prior to substitution. Preferably the substitutions areconservative substitutions. An example of such a motif can be foundwithin the CDR2 region of the 22H8 anti-IL-23/IL-12 antibody. The motifmay be described by the following formula:

an amino acid sequence sequence WX¹KG, wherein X1 is alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine or tryptophan,and preferably is alanine or valine;

Other examples of common motifs found with the CDRs anti-IL-23/IL-12antibodies of the present invention include:

a motif in the CDR3 region comprising the amino acid sequenceYAYX¹GDAFDP, wherein X¹ is alanine or isoleucine; (SEQ ID NO. 339)and/ora motif in the CDR3 region comprising the amino acid sequence SDYFNX¹,wherein X¹ is isoleucine or valine; (SEQ ID NO. 340)and/ora motif in the CDR4 region comprising the amino acid sequence QX¹SQX²,whereinX¹ is alanine or serine, andX² is selected from the group consisting of glycine, asparagine,glutamine, cysteine, serine, threonine, and tyrosine;preferably X² is serine or threonine;and/ora motif in the CDR5 region comprising the amino acid sequence ASX¹LA,wherein X¹ is lysine or threonine; (SEQ ID NO. 341)and/ora motif in the CDR6 region comprising the amino acid sequenceQSYYDX¹NAGYG, wherein X¹ is alanine or valine. (SEQ ID NO. 342)

Examples of common motifs found with the CDRs of the IL-23 antibodies ofthe present invention include:

a motif in the CDR2 region comprising the amino acid sequenceYYAX¹WAX²G, whereinX¹ is selected from the group consisting of serine, proline andaspartate, andX² is selected from the group consisting of lysine and glutamine; (SEQID NO. 337) and/ora motif in the CDR5 region comprising the amino acid sequence AX¹TLX²S,whereinX¹ is selected from the group consisting of serine and alanineX² is selected from the group consisting of alanine and threonine. (SEQID NO. 338)

Examples of common motifs found with the CDRs of the IL-6 antibodies ofthe present invention include:

a motif in the CDR2 region comprising the amino acid sequenceYIYTDX¹STX²YANWAKG, whereinX¹ is selected from the group consisting of glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine; andX² is selected from the group consisting of phenylalanine, tryptophan,and tyrosine; and preferably X¹ is serine or threonine and X2 istryptophan or tyrosine; (SEQ ID NO. 335) and/ora motif in the CDR5 region comprising the amino acid sequence RX¹STLX²S,wherein X¹ and X² are independently alanine or threonine. (SEQ ID NO.336)

Modification to Incorporate Non-Natural Amino Acids

The present invention provides for the incorporation of non-naturalamino acid residues into the anti-IL-6 anti-IL-23 and anti-IL-23/IL-12antibodies, or derivatives thereof, to provide a point of the attachmentfor the anti-IL-6 antibodies, or derivatives thereof, to anti-IL-23 oranti-IL-23/IL-12 antibodies, or derivatives thereof. The person skilledin the art will be aware of a number of potentially suitable non-naturalamino acids, including, for instance azidohomoalanine (Aha). Additionalnon natural amino acids include azidonorleucine, 3-(1-naphthyl)alanine,3-(2-naphthyl)alanine, p-ethynyl-phenylalanine,p-propargly-oxy-phenylalanine, m-ethynyl-phenylalanine,6-ethynyl-tryptophan, 5-ethynyl-tryptophan,(R)-2-amino-3-(4-ethynyl-1H-pyrol-3-yl)propanic acid,p-bromophenylalanine, p-idiophenylalanine, p-azidophenylalanine,3-(6-chloroindolyl)alanine, 3-(6-bromoindoyl)alanine,3-(5-bromoindolyl)alanine, homoallylglycine, homopropargylglycine, andp-chlorophenylalanine. In a preferred embodiment, the non-natural aminoacid is Aha.

The person skilled in the art will also appreciate that in order tocontrol the site of attachment it is necessary to engineer the aminoacid sequences of the antibodies or derivatives thereof, such that therenon-natural amino acids are only located in positions where attachmentis to occur. In an embodiment a non-natural amino acid may be located atthe N-terminus of an antibody, or derivative thereof, as disclosedherein. In an embodiment a non-natural amino acid may be located at theC-terminus of an antibody, or derivative thereof, as disclosed herein.In an embodiment the non-natural amino acid may be located in the linkerregion between the VH and VL portions of an scFv as disclosed herein(e.g. within SEQ ID NO. 327). In an embodiment there is a single pointof attachment in each antibody to be incorporated into the bivalent,bispecific construct. Examples of antibodies, or derivatives thereof,scFvs, and/or portions of the bivalent bispecific constructs of thepresent invention include SEQ ID No. 287 to 312.

In an embodiment the incorporation of non-natural amino acids isachieved by expressing the antibodies in auxotrophic host cells thatincorporate a non-natural amino acid (such as Aha) in place ofmethionine (Met). In order for there to be a single site of attachmentthe antibody nucleotide sequences must be engineered to remove anynaturally occurring codons for methionine not located at the desiredsite of attachment. This may be achieved by substituting them withcodons for other amino acids (typically natural amino acids). Since 1-2methionine residues are frequently found within framework regions andCDRs of immunoglobulin VH-regions, and infrequently in VL regions, it isnecessary to find suitable replacements for these residues where theyoccur without impacting the expression, stability or function (e.g.binding or target neutralising activity) of the desired protein. Thismethionine-free scFv can then be optimized for expression in amethionine auxotrophic bacterial strain, purified, refolded and testedfor biologic activity. Optionally more than one methionine codon can beleft in the sequence to allow for incorporation of more than onenon-natural amino acid (such as Aha).

If a methionine is not naturally present at the desired site ofattachment a single (or optionally, more than one) methionine codon canbe introduced that serves as an insertion site for a non-natural aminoacid with a chemically reactive site for attachment.

The antibodies modified to include non-natural amino acids may beattached to one or more separate entities. These entities include linkergroups and/or other similarly modified antibodies. Examples of suitablelinkers are known in the art and include short peptide sequences. Thepresent invention also provides for the use of PEG as a linker. Thus, inan embodiment an anti-IL-6 antibody, or derivative thereof,incorporating a non-natural amino acid may be covalently linked to a PEGlinker group, which PEG linker group is in turn attached to ananti-IL-23 or anti-IL-23/IL-12 antibody, or derivative thereof,incorporating a non-natural amino acid. Such bi-specific, PEGylatedconstructs can then be purified and refolded to yield a stable,biologically active therapeutic protein.

Suitably, the antibodies or derivatives thereof in the present inventionmodified to include non-natural aminoacids may directly (e.g. withoutthe use of linker groups) be linked to other similarly modifiedmolecules, including but not limited to, other antibodies or derivativesthereof, dyes, drugs or toxins.

Labelling and Derivatization

A bivalent bispecific construct or antibody of the invention can bederivatized or linked to another molecule. In general, the bivalentbispecific construct is derivatized such that binding and biologicalactivity of the constituent antibodies or derivatives thereof is notaffected adversely by the derivatization or labelling.

For example, a bivalent bispecific construct of the invention can befunctionally linked (by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other molecular entities, suchas a detection agent, a cytotoxic agent, a pharmaceutical agent, and/ora protein or peptide that can mediate association of the antibody orantibody portion with another molecule (such as a streptavidin coreregion or a polyhistidine tag).

A type of derivatized bivalent bispecific construct is a labelledbivalent bispecific construct. Useful detection agents with whichbivalent bispecific construct of the invention may be derivatizedinclude fluorescent compounds, including fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonylchloride, phycoerythrin, lanthanide phosphors and the like. An bivalentbispecific construct y may also be labelled with enzymes that are usefulfor detection, such as horseradish peroxidase, -galactosidase,luciferase, alkaline phosphatase, glucose oxidase and the like. When abivalent bispecific construct is labelled with a detectable enzyme, itis detected by adding additional reagents that the enzyme uses toproduce a reaction product that can be discerned.

For example, when the agent horseradish peroxidase is present, theaddition of hydrogen peroxide and diaminobenzidine leads to a colouredreaction product, which is detectable. A bivalent bispecific constructmay also be labelled with biotin, and detected through indirectmeasurement of avidin or streptavidin binding. A bivalent bispecificconstruct may also be labelled with a predetermined polypeptide epitopesrecognized by a secondary reporter (e.g. leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags). In some embodiments, labels are attached by spacer arms ofvarious lengths to reduce potential steric hindrance.

The bivalent bispecific construct may also be labelled with aradiolabelled amino acid. The radiolabel may be used for both diagnosticand therapeutic purposes. The radio-labelled bivalent bispecificconstruct may be used diagnostically, for example, for determining IL-6and/or IL-23 levels in a subject. Further, the radio-labelled bivalentbispecific construct may be used therapeutically for treating diseasesmediated by the T_(H)17 pathway. Examples of radiolabels include, butare not limited to, the following radioisotopes or radionuclides-3H,14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I. Radioisotopes may also bebound to the antibody or bispecific by derivitization with a chelationmoiety such as DOTA. Several of the useful imaging and therapeuticradioisotopes bind tightly to these chelators.

A bivalent bispecific construct may also be derivatized with a chemicalgroup such as polyethylene glycol (PEG), a methyl or ethyl group, or acarbohydrate group. These groups may be useful to improve the biologicalcharacteristics of the antibody, e.g. to increase serum half-life or toincrease tissue binding.

Expression of Antibodies/Derivatives

The bivalent, bispecific constructs of the present invention, and theantibodies, and derivatives thereof, that make up the bivalent,bispecific constructs can be expressed using conventional recombinanttechnology. In addition where the constructs and/or antibodies, andderivatives thereof, comprise non-natural amino acids, recombinantmethods as described in WO 2007/130453 may be used. The nucleotidesequences, vectors, host cells etc. used to express the bivalentbispecific constructs and the antibodies, and derivatives thereof areobjects of the present invention

Polynucleotides

In an embodiment, the present invention provides nucleotide sequencesencoding the bivalent, bispecific constructs and the antibodies, andderivatives thereof, that make up the bivalent, bispecific constructs asdefined above.

Thus the present invention encompasses nucleotide sequences encoding (1)monoclonal antibodies according to the invention (2) humanizedantibodies according to the invention (3) variant antibodies based on(1) & (2) according to the invention (4) derivatives of the antibodiesof (1) to (3) according to the invention, and (5) bivalent, bispecificconstructs according to the invention.

In an embodiment the nucleotide sequences encode portions of anti-IL-6antibodies. Examples of such sequences are given in SEQ ID NOs. 7 and 9,which are the nucleotide sequences of the VH and VL regions of the IL-6antibody designated 13A8.

In an embodiment the nucleotide sequences encode portions of anti-IL-23antibodies. Examples of such sequences are given in SEQ ID NOs. 87 and89, which are the nucleotide sequences of the VH and VL regions of theanti-IL-23 antibody designated 31A12.

In an embodiment the nucleotide sequences encode portions ofanti-IL-23/IL-12 antibodies. Examples of such sequences are given in SEQID NOs. 137 and 139, which are the nucleotide sequences of the VH and VLregions of the anti-IL-23/IL-12 antibody designated 22H8.

In an embodiment the bivalent, bispecific construct may be expressed asa single product.

Promoters

In an embodiment the nucleotide sequences of the present invention areoperably linked to a promoter sequence. Examples of suitable promotersinclude, but are not limited to, T5/Lac promoter: T7/Lac or modifiedT7/lac promoters, Trc or tac promoters, phage pL or pR temperatureinducible promoters, tetA promoter/operator, araBAD (pBAD) promoter,rhaPBAD promoter and lac UV5 promoter. Other suitable promoters may beidentified from Terpe, K. (2006) (Appl Microbiol Biotechnol 72:211-222).In a preferred embodiment the promoter is a T5/Lac promoter.

Vectors

In an embodiment the present invention provides a vector comprising anucleotide e sequence of the present invention optionally, operablylinked to a promoter sequence.

Host Cells

In an embodiment the present invention provides a host cell transfectedwith a vector of the present invention and capable of expressing thenucleotide sequences contained within the vectors. Optionally, the hostcell is an auxotrophic cell, capable of incorporating a non-naturalamino acid in place of a particular natural amino acid (e.g. AHA inplace of Met). The host cell may be a prokaryotic cell or an eukaryoticcell. Suitable eukaryotic cells include yeast cells, mammalian cells andinsect cells. Preferably the host cells are prokaryotic, in particular,E. coli B384 which are methionine auxotrophic cells. Alternatively, thecells are mammalian cells, more preferably they are human cells, yetmore preferably they are human embryonic kidney cells (e.g. HEK293 orHEK 293c18 cells) or CHO cells.

Primers

In an embodiment of the invention primers for the cloning and expressionof the anti-IL-6, anti-IL-23 antibodies and anti-IL-23/IL-12 antibodies,and derivatives thereof, are provided. These primers vary in lengthbetween 10 and 40 nucleotides, preferably they are between 15 and 30nucleotides in length. The person skilled in the art will be able todetermine suitable primer sequences given the disclosure of the nucleicacid sequences of the antibodies, and derivatives thereof, disclosedherein. Particular primer sequences of interest are given in SEQ ID NOs0.200-258, which are useful for the cloning and expression of theantibodies and scFvs disclosed herein.

Incorporation of Non-Natural Amino Acids

The use of non-natural amino acids to allow for conjugating moieties topeptides is disclosed in WO 2007/130453. Such protein engineering isalso discussed below.

The first step in the protein engineering process is usually to select aset of non-natural amino acids that have the desired chemicalproperties. The selection of non-natural amino acids depends onpre-determined chemical properties and the modifications one would liketo make in the target molecule or target protein. Non-natural aminoacids, once selected, can either be purchased from vendors, orchemically synthesized. Any number of non-natural amino acids may beincorporated into the target molecule and may vary according to thenumber of desired chemical moieties that are to be attached. Thechemical moieties may be attached to all or only some of the non-naturalamino acids. Further, the same or different non-natural amino acids maybe incorporated into the molecule, depending on the desired outcome. Incertain embodiments, at least two different non-natural amino acids areincorporated into the molecule and one chemical moiety, such as PEG, isattached to one of the non-natural amino acid residues, while anotherchemical moiety, such as a cytotoxic agent, is attached to the othernon-natural amino acid.

A wide variety of non-natural amino acids can be used in the methods ofthe invention. Typically, the non-natural amino acids of use in theinvention are selected or designed to provide additional characteristicsunavailable in the twenty natural amino acids. For example, non-naturalamino acids are optionally designed or selected to modify the biologicalproperties of a molecule, including a protein, e.g., into which they areincorporated. For example, the following properties are optionallymodified by inclusion of an non-natural amino acid into a molecule, suchas a protein: toxicity, biodistribution, solubility, stability, e.g.,thermal, hydrolytic, oxidative, resistance to enzymatic degradation, andthe like, facility of purification and processing, structuralproperties, spectroscopic properties, chemical and/or photochemicalproperties, catalytic activity, ability to function as a vaccine, redoxpotential, half-life, ability to react with other molecules, e.g.,covalently or noncovalently, and the like.

As used herein an “non-natural amino acid” refers to any amino acid,modified amino acid, or amino acid analogue other than selenocysteineand the following twenty genetically encoded alpha-amino acids: alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine.The generic structure of an alpha-amino acid is illustrated by FormulaI:

A non-natural amino acid is typically any structure having Formula Iwherein the R group is any substituent other than one used in the twentynatural amino acids. See, e.g., any biochemistry text such asBiochemistry by L. Stryer, 3rd ed. 1988, Freeman and Company, New York,for structures of the twenty natural amino acids. Note that thenon-natural amino acids disclosed herein may be naturally occurringcompounds other than the twenty alpha-amino acids above. Because thenon-natural amino acids disclosed herein typically differ from thenatural amino acids in side chain only, the non-natural amino acids formamide bonds with other amino acids, e.g., natural or non-natural, in thesame manner in which they are formed in naturally occurring proteins.However, the non-natural amino acids have side chain groups thatdistinguish them from the natural amino acids. For example, R in FormulaI optionally comprises an alkyl-, aryl-, aryl halide, vinyl halide,beta-silyl alkenyl halide, beta-silyl alkenyl sulfonates, alkyl halide,acetyl, ketone, aziridine, nitrile, nitro, nitrile oxide, halide, acyl-,keto-, azido-, ketal, acetal, hydroxyl-, hydrazine, cyano-, halo-,hydrazide, alkenyl, alkynyl, ether, thioether, epoxide, sulfone, boronicacid, boronate ester, borane, phenylboronic acid, thiol, seleno-,sulfonyl-, borate, boronate, phospho, phosphono, phosphine,heterocyclic-, pyridyl, naphthyl, benzophenone, a constrained ring suchas cyclooctyne, cyclopropene, norbornene thioester, enone, imine,aldehyde, ester, thioacid, hydroxylamine, amino, carboxylic acid,alpha-keto carboxylic acid, alpha or beta unsaturated acids and amides,glyoxyl amide, or organosilane, pyrones, tetrazine, pyridazine,hydrzaides, hydrazines, alkoxyamines, aryl sulfonates, aryl halides,thiosemicarbazide, semicarbazide, tetrazole group or the like or anycombination thereof.

Specific examples of unnatural amino acids include, but are not limitedto, p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-GlcNAc.beta.-serine, .beta.-O-GlcNAc-L-serine, atri-O-acetyl-GalNAc-.alpha.-threonine, an .alpha.-GalNAc-L-threonine, anL-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, ap-azido-L-phenylalanine, a p-acyl-L-phenylalanine, ap-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, aphosphonotyrosine, a p-iodo-phenylalanine, a p-bromophenylalanine, ap-amino-L-phenylalanine, an isopropyl-L-phenylalanine, pyrrolysine,N-sigma-o-azidobenzyloxycarbonyl-L-Lysine (AzZLys),N-sigma-propargyloxycarbonyl-L-Lysine,N-sigma-2-azidoethoxycarbonyl-L-Lysine,N-sigma-tert-butyloxycarbonyl-L-Lysine (BocLys),N-sigma-allyloxycarbonyl-L-Lysine (AlocLys), N-sigma-acetyl-L-Lysine(AcLys), N-sigma-benzyloxycarbonyl-L-Lysine (ZLys),N-sigma-cyclopentyloxycarbonyl-L-Lysine (CycLys),N-sigma-D-prolyl-L-Lysine, N-sigma-nicotinoyl-L-Lysine (NicLys),N-sigma-N-Me-anthraniloyl-L-Lysine (NmaLys), N-sigma-biotinyl-L-Lysine,N-sigma-9-fluorenylmethoxycarbonyl-L-Lysine, N-sigma-methyl-L-Lysine,N-sigma-dimethyl-L-Lysine, N-sigma-trimethyl-L-Lysine,N-sigma-isopropyl-L-Lysine, N-sigma-dansyl-L-Lysine,N-sigma-o,p-dinitrophenyl-L-Lysine, N-sigma-p-toluenesulfonyl-L-Lysine,N-sigma-DL-2-amino-2-carboxyethyl-L-Lysine,N-sigma-phenylpyruvamide-L-Lysine, N-sigma-pyruvamide-L-Lysine. thoselisted below, or elsewhere herein, and the like; and for example areselected from p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-GlcNAc.beta.-serine, .beta.-O-GlcNAc-L-serine, atri-O-acetyl-GalNAc-.alpha.-threonine, an .alpha.-GalNAc-L-threonine, anL-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, ap-azido-L-phenylalanine, a p-acyl-L-phenylalanine, ap-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, aphosphonotyrosine, a p-iodo-phenylalanine, a p-bromophenylalanine, ap-amino-L-phenylalanine and an isopropyl-L-phenylalanine.

Aryl substitutions may occur at various positions, e.g. ortho, meta,para, and with one or more functional groups placed on the aryl ring.Other non-natural amino acids of interest include, but are not limitedto, amino acids comprising a photoactivatable cross-linker, spin-labeledamino acids, dye-labeled amino acids, fluorescent amino acids, metalbinding amino acids, metal-containing amino acids, radioactive aminoacids, amino acids with novel functional groups, amino acids withaltered hydrophilicity, hydrophobocity, polarity, or ability to hydrogenbond, amino acids that covalently or noncovalently interact with othermolecules, photocaged and/or photoisomerizable amino acids, amino acidscomprising biotin or a biotin analogue, glycosylated amino acids such asa sugar substituted serine, other carbohydrate modified amino acids,keto containing amino acids, amino acids comprising polyethylene glycolor a polyether, a polyalcohol, or a polysaccharide, amino acids that canundergo metathesis, amino acids that can undergo cycloadditions, heavyatom substituted amino acids, chemically cleavable and/or photocleavableamino acids, amino acids with an elongated side chains as compared tonatural amino acids, e.g., polyethers or long chain hydrocarbons, e.g.,greater than about 5 or greater than about 10 carbons, carbon-linkedsugar-containing amino acids, redox-active amino acids, amino thioacidcontaining amino acids, amino acids containing a drug moiety, and aminoacids comprising one or more toxic moieties.

In addition to non-natural amino acids that contain novel side chains,non-natural amino acids also optionally comprise modified backbonestructures, e.g., as illustrated by the structures of Formula II andIII:

wherein Z typically comprises OH, NH.sub.2, SH, NH.sub.20-, NH—R′,R′NH—, R′S—, or S—R′—; X and Y, which may be the same or different,typically comprise S, N, or O, and R and R′, which are optionally thesame or different, are typically selected from the same list ofconstituents for the R group described above for the non-natural aminoacids having Formula I as well as hydrogen or (CH.sub.2).sub.x or thenatural amino acid side chains. For example, non-natural amino acidsdisclosed herein optionally comprise substitutions in the amino orcarboxyl group as illustrated by Formulas II and III. Non-natural aminoacids of this type include, but are not limited to, .alpha.-hydroxyacids, .alpha.-thioacids .alpha.-aminothiocarboxylates, or.alpha.-.alpha.-disubstituted amino acids, with side chainscorresponding e.g. to the twenty natural amino acids or to non-naturalside chains. They also include but are not limited to .beta.-amino acidsor .gamma.-amino acids, such as substituted .beta.-alanine and.gamma.-amino butyric acid. In addition, substitutions or modificationsat the .alpha.-carbon optionally include L or D isomers, such asD-glutamate, D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and thelike. Other structural alternatives include cyclic amino acids, such asproline analogs as well as 3-, 4-, 6-, 7-, 8-, and 9-membered ringproline analogs. Some non-natural amino acids, such as aryl halides(p-bromo-phenylalanine, p-iodophenylalanine, provide versatile palladiumcatalyzed cross-coupling reactions with ethyne or acetylene reactionsthat allow for formation of carbon-carbon, carbon-nitrogen andcarbon-oxygen bonds between aryl halides and a wide variety of couplingpartners.

For example, many non-natural amino acids are based on natural aminoacids, such as tyrosine, glutamine, phenylalanine, and the like.Tyrosine analogs include para-substituted tyrosines, ortho-substitutedtyrosines, and meta substituted tyrosines, wherein the substitutedtyrosine comprises an acetyl group, a benzoyl group, an amino group, ahydrazine, an hydroxyamine, a thiol group, a carboxy group, an isopropylgroup, a methyl group, a C6-C20 straight chain or branched hydrocarbon,a saturated or unsaturated hydrocarbon, an O-methyl group, a polyethergroup, a nitro group, or the like. In addition, multiply substitutedaryl rings are also contemplated. Glutamine analogs include, but are notlimited to, .alpha.-hydroxy derivatives, .beta.-substituted derivatives,cyclic derivatives, and amide substituted glutamine derivatives.Exemplary phenylalanine analogs include, but are not limited to,meta-substituted phenylalanines, wherein the substituent comprises ahydroxy group, a methoxy group, a methyl group, an allyl group, anacetyl group, or the like.

Specific examples of non-natural amino acids include, but are notlimited to, o, m and/or p forms of amino acids or amino acid analogs(non-natural amino acids), including homoallylglycine, cis- ortrans-crotylglycine, 6,6,6-trifluoro-2-aminohexanoic acid,2-aminopheptanoic acid, norvaline, norleucine, O-methyl-L-tyrosine, o-,m-, or p-methyl-phenylalanine, O-4-allyl-L-tyrosine, a4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAc.beta.-serine, an L-Dopa, afluorinated phenylalanine, an isopropyl-L-phenylalanine, ap-azidophenylalanine, a p-acyl-L-phenylalanine, ap-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, aphosphonotyrosine, a p-iodo-phenylalanine, o-, m-, orp-bromophenylalanine, 2-, 3-, or 4-pyridylalanine, p-idiophenylalanine,diaminobutyric acid, aminobutyric acid, benzofuranylalanine,3-bromo-tyrosine, 3-(6-chloroindolyl)alanine, 3-(6-bromoindolyl)alanine,3-(5-bromonindolyl)alanine, p-chlorophenylalanine,p-ethynyl-phenylalanine, p-propargly-oxy-phenylalanine,m-ethynyl-phenylalanine, 6-ethynyl-tryptophan, 5-ethynyl-tryptophan,(R)-2-amino-3-(4-ethynyl-1H-pyrol-3-yl)propanoic acid, azidonorleucine,azidohomoalanine, p-acetylphenylalanine, p-amino-L-phenylalanine,homoproparglyglycine, p-ethyl-phenylalanine, p-ethynyl-phenylalanine,p-propargly-oxy-phenylalanine, isopropyl-L-phenylalanine, an3-(2-naphthyl)alanine, 3-(1-naphthyl)alanine, 3-idio-tyrosine,O-propargyl-tyrosine, homoglutamine, an O-4-allyl-L-tyrosine, a4-propyl-L-tyrosine, a 3-nitro-L-tyrosine, atri-O-acetyl-GlcNAc.beta.-serine, an L-Dopa, a fluorinatedphenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine,a p-acyl-L-phenylalanine, a p-acetyl-L-phenylalanine, anm-acetyl-L-phenylalanine, selenomethionine, telluromethionine,selenocysteine, an alkyne phenylalanine, an O-allyl-L-tyrosine, anO-(2-propynyl)-L-tyrosine, a p-ethylthiocarbonyl-L-phenylalanine, ap-(3-oxobutanoyl)-L-phenylalanine, a p-benzoyl-L-phenylalanine, anL-phosphoserine, a phosphonoserine, a phosphonotyrosine,homoproparglyglycine, azidohomoalanine, a p-iodo-phenylalanine, ap-bromo-L-phenylalanine, dihydroxy-phenylalanine,dihydroxyl-L-phenylalanine, a p-nitro-L-phenylalanine, anm-methoxy-L-phenylalanine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, and anisopropyl-L-phenylalanine, trifluoroleucine, norleucine, 4-, 5-, or6-fluoro-tryptophan, 4-aminotryptophan, 5-hydroxytryptophan, biocytin,aminooxyacetic acid, m-hydroxyphenylalanine, m-allyl phenylalanine,m-methoxyphenylalanine group, .beta.-GlcNAc-serine,.alpha.-GalNAc-threonine, p-acetoacetylphenylalanine,para-halo-phenylalanine, seleno-methionine, ethionine,S-nitroso-homocysteine, thia-proline, 3-thienyl-alanine,homo-allyl-glycine, trifluoroisoleucine, trans andcis-2-amino-4-hexenoic acid, 2-butynyl-glycine, allyl-glycine,para-azidophenylalanine, para-cyano-phenylalanine,para-ethynyl-phenylalanine, hexafluoroleucine, 1,2,4-triazole-3-alanine,2-fluoro-histidine, L-methyl histidine, 3-methyl-L-histidine,.beta.-2-thienyl-L-alanine, .beta.-(2-thiazolyl)-DL-alanine,homoproparglyglycine (HPG) and azidohomoalanine (AHA) and the like. Thestructures of a variety of non-limiting non-natural amino acids areprovided in the figures, e.g., FIGS. 29, 30, and 31 of US 2003/0108885A1, the entire content of which is incorporated herein by reference.

Tyrosine analogs include para-substituted tyrosines, ortho-substitutedtyrosines, and meta substituted tyrosines, wherein the substitutedtyrosine comprises an acetyl group, a benzoyl group, an amino group, ahydrazine, an hydroxyamine, a thiol group, a carboxy group, an isopropylgroup, a methyl group, a C6-C20 straight chain or branched hydrocarbon,a saturated or unsaturated hydrocarbon, an O-methyl group, a polyethergroup, a nitro group, or the like. In addition, multiply substitutedaryl rings are also contemplated. Glutamine analogs of the inventioninclude, but are not limited to, .alpha.-hydroxy derivatives,.beta.-substituted derivatives, cyclic derivatives, and amidesubstituted glutamine derivatives. Example phenylalanine analogsinclude, but are not limited to, meta-substituted phenylalanines,wherein the substituent comprises a hydroxy group, a methoxy group, amethyl group, an allyl group, an acetyl group, or the like. Lysineanalogs include N-sigma substituted such as pyrrolysine,N-sigma-o-azidobenzyloxycarbonyl-L-Lysine (AzZLys),N-sigma-propargyloxycarbonyl-L-Lysine,N-sigma-2-azidoethoxycarbonyl-L-Lysine,N-sigma-tert-butyloxycarbonyl-L-Lysine (BocLys),N-sigma-allyloxycarbonyl-L-Lysine (AlocLys), N-sigma-acetyl-L-Lysine(AcLys), N-sigma-benzyloxycarbonyl-L-Lysine (ZLys),N-sigma-cyclopentyloxycarbonyl-L-Lysine (CycLys),N-sigma-D-prolyl-L-Lysine, N-sigma-nicotinoyl-L-Lysine (NicLys),N-sigma-N-Me-anthraniloyl-L-Lysine (NmaLys), N-sigma-biotinyl-L-Lysine,N-sigma-9-fluorenylmethoxycarbonyl-L-Lysine, N-sigma-methyl-L-Lysine,N-sigma-dimethyl-L-Lysine, N-sigma-trimethyl-L-Lysine,N-sigma-isopropyl-L-Lysine, N-sigma-dansyl-L-Lysine,N-sigma-o,p-dinitrophenyl-L-Lysine, N-sigma-p-toluenesulfonyl-L-Lysine,N-sigma-DL-2-amino-2carboxyethyl-L-Lysine,N-sigma-phenylpyruvamide-L-Lysine, N-sigma-pyruvamide-L-Lysine

Additionally, other examples optionally include (but are not limited to)an non-natural analog of a tyrosine amino acid; an non-natural analog ofa glutamine amino acid; an non-natural analog of a phenylalanine aminoacid; an non-natural analog of a serine amino acid; an non-naturalanalog of a threonine amino acid; an alkyl, aryl, acyl, azido, cyano,halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol,sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono,phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto,ketal, acetal, strained cyclooctyne, strained cycloalkene, cyclopropene,norbornenes, nitrile oxides, beta-silyl alkenyl halide, beta-silylalkenyl sulfonates, pyrones, tetrazine, pyridazine, alkoxyamines, arylsulfonates, aryl halides, thiosemicarbazide, semicarbazide, tetrazole,alpha-ketoacid or amino substituted amino acid, or any combinationthereof; an amino acid with a photoactivatable cross-linker; aspin-labeled amino acid; a fluorescent amino acid; an amino acid with anovel functional group; an amino acid that covalently or noncovalentlyinteracts with another molecule; a metal binding amino acid; ametal-containing amino acid; a radioactive amino acid; a photocagedamino acid; a photoisomerizable amino acid; a biotin or biotin-analogcontaining amino acid; a glycosylated or carbohydrate modified aminoacid; a keto containing amino acid; an amino acid comprisingpolyethylene glycol; an amino acid comprising polyether; a heavy atomsubstituted amino acid; a chemically cleavable or photocleavable aminoacid; an amino acid with an elongated side chain; an amino acidcontaining a toxic group; a sugar substituted amino acid, e.g., a sugarsubstituted serine or the like; a carbon-linked sugar-containing aminoacid; a redox-active amino acid; an .alpha.-hydroxy containing acid; anamino thio acid containing amino acid; an .alpha.,.alpha. disubstitutedamino acid; a .beta.-amino acid; and a cyclic amino acid.

Typically, the non-natural amino acids utilized herein for certainembodiments may be selected or designed to provide additionalcharacteristics unavailable in the twenty natural amino acids. Forexample, non-natural amino acid are optionally designed or selected tomodify the biological properties of a protein, e.g., into which they areincorporated. For example, the following properties are optionallymodified by inclusion of an non-natural amino acid into a protein:toxicity, biodistribution, solubility, stability, e.g., thermal,hydrolytic, oxidative, resistance to enzymatic degradation, and thelike, facility of purification and processing, structural properties,spectroscopic properties, chemical and/or photochemical properties,catalytic activity, redox potential, half-life, ability to react withother molecules, e.g., covalently or noncovalently, and the like.

Other examples of amino acid analogs optionally include (but are notlimited to) an non-natural analog of a tyrosine amino acid; annon-natural analog of a glutamine amino acid; an non-natural analog of aphenylalanine amino acid; an non-natural analog of a serine amino acid;an non-natural analog of a threonine amino acid; an alkyl, aryl, acyl,azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl,ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate,phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde,hydroxylamine, keto, ketal, acetal, strained cyclooctyne, strainedcycloalkene, cyclopropene, norbornenes, nitrile oxides, beta-silylalkenyl halide, beta-silyl alkenyl sulfonates, pyrones, tetrazine,pyridazine, alkoxyamines, aryl sulfonates, aryl halides,thiosemicarbazide, semicarbazide, tetrazole, alpha-ketoacid or aminosubstituted amino acid, or any combination thereof; an amino acid with aphotoactivatable cross-linker; a spin-labeled amino acid; a fluorescentamino acid; an amino acid with a novel functional group; an amino acidthat covalently or noncovalently interacts with another molecule; ametal binding amino acid; a metal-containing amino acid; a radioactiveamino acid; a photocaged amino acid; a photoisomerizable amino acid; abiotin or biotin-analogue containing amino acid; a glycosylated orcarbohydrate modified amino acid; a keto containing amino acid; an aminoacid comprising polyethylene glycol; an amino acid comprising polyether;a heavy atom substituted amino acid; a chemically cleavable orphotocleavable amino acid; an amino acid with an elongated side chain;an amino acid containing a toxic group; a sugar substituted amino acid,e.g., a sugar substituted serine or the like; a carbon-linkedsugar-containing amino acid; a redox-active amino acid; an.alpha.-hydroxy containing acid; an amino thio acid containing aminoacid; an .alpha.,.alpha. disubstituted amino acid; a .beta.-amino acid;and a cyclic amino acid other than proline.

Non-natural amino acids suitable for use in the methods of the inventionalso include those that have a saccharide moiety attached to the aminoacid side chain. In one embodiment, an non-natural amino acid with asaccharide moiety includes a serine or threonine amino acid with a Man,GalNAc, Glc, Fuc, or Gal moiety. Examples of non-natural amino acidsthat include a saccharide moiety include, but are not limited to, e.g.,a tri-O-acetyl-GlcNAc.beta.-serine, a .beta.-O-GlcNAc-L-serine, atri-O-acetyl-GalNAc-.alpha.-threonine, an .alpha.-GalNAc-L-threonine, anO-Man-L-serine, a tetra-acetyl-O-Man-L-serine, an O-GalNAc-L-serine, atri-acetyl-O-GalNAc-L-serine, a Glc-L-serine, atetraacetyl-Glc-L-serine, a fuc-L-serine, a tri-acetyl-fuc-L-serine, anO-Gal-L-serine, a tetra-acetyl-O-Gal-L-serine, a.beta.-O-GlcNAc-L-threonine, a tri-acetyl-.beta.-GlcNAc-L-threonine, anO-Man-L-threonine, a tetra-acetyl-O-Man-L-threonine, anO-GalNAc-L-threonine, a tri-acetyl-O-GalNAc-L-threonine, aGlc-L-threonine, a tetraacetyl-Glc-L-threonine, a fuc-L-threonine, atri-acetyl-fuc-L-threonine, an O-Gal-L-threonine, atetra-acetyl-O-Gal-L-serine, a .beta.-N-acetylglucosamine-O-serine,.alpha.-N-acetylgalactosamine-O-threonine, fluorescent amino acids suchas those containing naphthyl or dansyl or 7-aminocoumarin or7-hydroxycoumarin side chains, photocleavable or photoisomerizable aminoacids such as those containing azobenzene or nitrobenzyl Cys, Ser or Tyrside chains, p-carboxy-methyl-L-phenylalanine, homoglutamine,2-aminooctanoic acid, p-azidophenylalanine, p-benzoylphenylalanine,p-acetylphenylalanine, m-acetylphenylalanine, 2,4-diaminobutyric acid(DAB) and the like. The invention includes unprotected and acetylatedforms of the above. (See also, for example, WO 03/031464 A2, entitled“Remodeling and Glycoconjugation of Peptides”; and, U.S. Pat. No.6,331,418, entitled “Saccharide Compositions, Methods and Apparatus fortheir synthesis;” Tang and Tirrell, J. Am. Chem. Soc. (2001) 123:11089-11090; and Tang et al., Angew. Chem. Int. Ed., (2001) 40:8, all ofwhich are incorporated herein by reference in their entireties).

Many of the non-natural amino acids provided above are commerciallyavailable, e.g., from Sigma Aldrich (USA). Those that are notcommercially available are optionally synthesized as provided in theexamples of US 2004/138106 A1 (incorporated herein by reference) orusing standard methods known to those of skill in the art. For organicsynthesis techniques, see, e.g., Organic Chemistry by Fessendon andFessendon, (1982, Second Edition, Willard Grant Press, Boston Mass.);Advanced Organic Chemistry by March (Third Edition, 1985, Wiley andSons, New York); and Advanced Organic Chemistry by Carey and Sundberg(Third Edition, Parts A and B, 1990, Plenum Press, New York), and WO02/085923, all of which are hereby incorporated by reference.

For example, meta-substituted phenylalanines are synthesized in aprocedure as outlined in WO 02/085923 (see, e.g., FIG. 14 of thepublication). Typically, NBS (N-bromosuccinimide) is added to ameta-substituted methylbenzene compound to give a meta-substitutedbenzyl bromide, which is then reacted with a malonate compound to givethe meta substituted phenylalanine. Typical substituents used for themeta position include, but are not limited to, ketones, methoxy groups,alkyls, acetyls, and the like. For example, 3-acetyl-phenylalanine ismade by reacting NBS with a solution of 3-methylacetophenone. For moredetails see the examples below. A similar synthesis is used to produce a3-methoxy phenylalanine. The R group on the meta position of the benzylbromide in that case is—OCH.sub.3. (See, e.g., Matsoukas et al., J. Med.Chem., 1995, 38, 4660-4669, incorporated by reference in its entirety).

In some cases, the design of non-natural amino acids is biased by knowninformation about the active sites of synthetases, e.g., external mutanttRNA synthetases used to aminoacylate an external mutant tRNA. Forexample, three classes of glutamine analogs are provided, includingderivatives substituted at the nitrogen of amide (1), a methyl group atthe .gamma.-position (2), and a N-Cy-cyclic derivative (3). Based uponthe x-ray crystal structure of E. coli GlnRS, in which the key bindingsite residues are homologous to yeast GlnRS, the analogs were designedto complement an array of side chain mutations of residues within a10.ANG. shell of the side chain of glutamine, e.g., a mutation of theactive site Phe233 to a small hydrophobic amino acid might becomplemented by increased steric bulk at the Cy position of Gln.

For example, N-phthaloyl-L-glutamic 1,5-anhydride (compound number 4 inFIG. 23 of WO 02/085923) is optionally used to synthesize glutamineanalogs with substituents at the nitrogen of the amide. (See, e.g., King& Kidd, J. Chem. Soc., 3315-3319, 1949; Friedman & Chatterrji, J. Am.Chem. Soc. 81, 3750-3752, 1959; Craig et al., J. Org. Chem. 53,1167-1170, 1988; and Azoulay et al., Eur. J. Med. Chem. 26, 201-5, 1991,all of which are hereby incorporated by reference in their entireties).The anhydride is typically prepared from glutamic acid by firstprotection of the amine as the phthalimide followed by refluxing inacetic acid. The anhydride is then opened with a number of amines,resulting in a range of substituents at the amide. Deprotection of thephthaloyl group with hydrazine affords a free amino acid as shown inFIG. 23 of WO 2002/085923.

Substitution at the .gamma.-position is typically accomplished viaalkylation of glutamic acid. (See, e.g., Koskinen & Rapoport, J. Org.Chem. 54, 1859-1866, 1989, hereby incorporated by reference). Aprotected amino acid, e.g., as illustrated by compound number 5 in FIG.24 of WO 02/085923, is optionally prepared by first alkylation of theamino moiety with 9-bromo-9-phenylfluorene (PhflBr) (see, e.g., Christie& Rapoport, J. Org. Chem. 1989, 1859-1866, 1985, hereby incorporated byreference) and then esterification of the acid moiety usingO-tert-butyl-N,N′-diisopropylisourea. Addition ofKN(Si(CH.sub.3).sub.3).sub.2 regioselectively deprotonates at the.alpha.-position of the methyl ester to form the enolate, which is thenoptionally alkylated with a range of alkyl iodides. Hydrolysis of thet-butyl ester and Phfl group gave the desired .gamma.-methyl glutamineanalog (Compound number 2 in FIG. 24 of WO 02/085923, herebyincorporated by reference).

An N-Cy cyclic analog, as illustrated by Compound number 3 in FIG. 25 ofWO 02/085923, is optionally prepared in 4 steps from Boc-Asp-Ot-Bu aspreviously described. (See, e.g., Barton et al., Tetrahedron Lett. 43,4297-4308, 1987, and Subasinghe et al., J. Med. Chem. 35 4602-7, 1992,each is hereby incorporated by reference). Generation of the anion ofthe N-t-Boc-pyrrolidinone, pyrrolidinone, or oxazolidone followed by theaddition of the compound 7, as shown in FIG. 25, results in a Michaeladdition product. Deprotection with TFA then results in the free aminoacids.

Trifluoroleucine (Tfl) and hexafluoroleucine (Hfl), may be synthesizedby various methods known in the art. For example,5′,5′,5′-trifluoro-DL-leucine may be synthesized in step-wise fashion byfirst diluting commercial trifluoromethyl crotonic acid with ethanol andhydrogenating it in the presence of a catalyst. Next, the mixture may berefluxed, and the ester distilled. Next,.alpha.-oximino-5′,5′,5′-trifluoroisocaproic acid may be derived byreflux and distillation, followed by recrystallization of5′,5′,5′-trifluoro-DL-leucine. Likewise,(S)-5,5,5,5′,5′,5′-Hexafluoroleucine may be prepared fromhexafluoroacetone and ethyl bromopyruvate in multiple steps, including ahighly enantioselective reduction of the carbonyl group in an.alpha.-keto ester by bakers' yeast or by catecholborane utilizing anoxazaborolidine catalyst. (For more details, see for example, Rennert,Anker, Biochem. 1963, 2, 471; Zhang, et al., Helv. Chim. Acta 1998, 81,174-181, R., Prot Sci. 7: 419-426 (1998); Hendrickson, et al., AnnualRev. Biochem. 73: 147-176 (2004); U.S. Patent Application Nos.20030108885 and 20030082575, as well as copending U.S. ProvisionalApplication No. 60/571,810, all of which are hereby incorporated byreference in their entireties). One point of novelty of the presentdisclosure relates to increased thermal and chemical stability ofleucine-zipper domain-rich molecules for which a fluorinated non-naturalamino acid(s) has been incorporated.

Likewise, homoproparglyglycine (HPG) and azidohomoalanine (AHA) may besynthesized by published methods. For example, according to Mangold, etal., Mutat. Res., 1989, 216, 27, which is hereby incorporated byreference in its entirety.

Synthesis of Bispecific Constructs General Methods of FormingBispecifics

In an embodiment bivalent bispecific constructs of the present inventionmay be made according the following method comprising:

-   -   (i) providing a host cell, the host cell comprising a vector        having a polynucleotide encoding an anti-IL-6 antibody, or        derivative thereof, which antibody or derivative is modified by        incorporation of at least one non-natural amino acid;    -   (ii) providing a host cell, the host cell comprising a vector        having a polynucleotide encoding an anti-IL-23 antibody, or        derivative thereof, which antibody or derivative is modified by        incorporation of at least one non-natural amino acid;    -   (iii) growing the host cells under conditions such that the host        cells express the modified anti-IL-6 antibody, or derivative        thereof, and the modified anti-IL-23 antibody, or derivative        thereof,    -   (iv) isolating the anti-IL-6 antibody, or derivative thereof,        and the anti-IL-23 antibody, or derivative thereof;    -   (v) reacting the anti-IL-6 antibody, or derivative thereof, with        the anti-IL-23 antibody, or derivative thereof, such that the        anti-IL-6 antibody, or derivative thereof, is coupled to the        anti-IL-23 antibody, or derivative thereof, through a linkage        between a non-natural amino acid of each portion.

Bispecific constructs of the present invention may also be made bymethods known in the art. These include somatic hybridization, chemicalcoupling and recombinant techniques

Somatic hybridization involves the fusion of two hybridomas andpurification of the bispecific secreted by the resulting quadromas. Twodifferent methods have been described: (1) fusion of two establishedhybridomas generates a quadroma (Milstein and Cuello, 1983; Suresh etal., 1986), and (2) fusion of one established hybridoma with lymphocytesderived from a mouse immunized with a second antigen generates trioma(Nolan and Kennedy, 1990). Somatic hybridization for development ofbsMAb involves methods similar to those for preparing hybridomas.However, the production and random association of two different heavychains and two different light chains within one cell leads to theassembly of a substantial proportion of non-functional molecules.Elaborate purification techniques need to be developed to purify thebispecific with the required specificity, and this mostly precludeslarge scale manufacture for clinical use. Nonetheless, the presentinvention provides a bivalent bispecific construct as disclosed abovemanufactured using somatic hybridization.

Chemical coupling of antibodies as known in the art was first carriedout nearly 40 years ago. The first bispecific polyclonal antibodies wereproduced by chemically coupling two different polyclonal antibodies(Nisonoff and Rivers, 1961). This chemical manipulation involved thedissociation of the two different antibodies at their inter Heavy chaindisulfide bonds, and cross linking of the two half molecules throughchemical conjugation. To prepare bsMAb, a large number of bifunctionalreagents reactive with ε-amino groups or hinge region thiol groups havebeen used. These cross-linkers can be classified into two categories,homo- and heterobifunctional reagents. Homobifunctional reagents reactwith the free thiols generated upon reduction of inter heavy chaindisulfide bonds. 5,5-Dithiobis(2-nitrobenzoic acid) (DTNB) oro-phenylenedimaleimide (O-PDM) can activate thiol groups on Fab′fragments of MAb. DTNB acts to regenerate disulfide bonds between thetwo Fabs, whereas O-PDM acts to form a thioether bond between the twoFab′. Generally, the thioether bond of O-PDM could be more stable thanthe disulfide bond regenerated by DTNB. Heterobifunctional reagents canintroduce a reactive group onto a protein that will enable it to reactwith a second protein. N-Succinimidyl-3-(2-pyridyldithio) propionate(SPDP) has been used to react with primary amino groups to introducefree thiol groups. SPDP can combine any two proteins that have exposedamino groups including antibodies and Fab′ fragments, regardless ofclass or isotype. However, this approach causes random cross-linking ofthe molecules, and hence exhibits batch to batch variations, andunwanted effects, such as, denaturation of the proteins, and/or loss ofantibody activity. Nonetheless, the present invention provides abivalent bispecific construct as disclosed above manufactured usingchemical coupling,

Recombinant techniques can also be used to make bispecific antibodies.Such bispecific antibodies derived by genetic engineering offer severaladvantages over conventional bispecific antibodies made by chemicalcross-linking or fusion of two hybridoma clones, including greatercontrol over the size, and affinity of the bispecific. By using only thevariable domains as building blocks, recombinant antibodies lack theFc-region of an antibody, and thus do not induce Fc-mediated immuneeffector function. A wide variety of different recombinant bispecificantibody formats have been developed over the past years. Amongst themtandem single-chain Fv molecules and diabodies and various derivativesthereof are the most widely used formats for the construction ofrecombinant bispecific antibodies. In one common theme, construction ofthese molecules starts from two single-chain Fv (scFv) fragments(variable regions of the immunoglobulin heavy and light chains linkedthrough a peptide linker) that recognize different antigens. Tandem scFvmolecules (taFv) represent a straightforward format simply connectingthe two scFv molecules with an additional peptide linker. The two scFvfragments present in these tandem scFv molecules form separate foldingentities. Thus various linkers can be used to connect the two scFvfragments and linkers with a length of up to 63 residues have beenreported. Although the parental scFv fragments can normally be expressedin soluble form in bacteria, it is, however, often observed that tandemscFv molecules form insoluble aggregates in bacteria. Hence, refoldingprotocols or the use of mammalian expression systems are routinelyapplied to produce soluble tandem scFv molecules. Thus present inventionprovides a bivalent bispecific construct as disclosed above manufacturedusing recombinant techniques as detailed above.

In a preferred method of manufacture as set out above the non-naturalamino acid contains an azide, cyano, nitrile oxides, alkyne, alkene,strained cyclooctyne, strained cycloalkene, cyclopropene, norbornenes oraryl, alkyl or vinyl halide, ketone, aldehyde, ketal, acetal, hydrazine,hydrazide, alkoxy amine, boronic acid, organotin, organosilicon,beta-silyl alkenyl halide, beta-silyl alkenyl sulfonates, pyrones,tetrazine, pyridazine, aryl sulfonates, thiosemicarbazide,semicarbazide, tetrazole, alpha-ketoacid group prior to linkage. Thenon-natural amino acid may be azidohomoalanine, homopropargylglycine,homoallylglycine, p-bromophenylalanine, p-iodophenylalanine,azidophenylalanine, acetylphenylalanine or ethynylephenylalanine, aminoacids containing an internal alkene such as trans-crotylalkene, serineallyl ether, allyl glycine, propargyl glycine, or vinyl glycine,pyrrolysine, N-sigma-o-azidobenzyloxycarbonyl-L-Lysine (AzZLys),N-sigma-propargyloxycarbonyl-L-Lysine,N-sigma-2-azidoethoxycarbonyl-L-Lysine,N-sigma-tert-butyloxycarbonyl-L-Lysine (BocLys),N-sigma-allyloxycarbonyl-L-Lysine (AlocLys), N-sigma-acetyl-L-Lysine(AcLys), N-sigma-benzyloxycarbonyl-L-Lysine (ZLys),N-sigma-cyclopentyloxycarbonyl-L-Lysine (CycLys),N-sigma-D-prolyl-L-Lysine, N-sigma-nicotinoyl-L-Lysine (NicLys),N-sigma-N-Me-anthraniloyl-L-Lysine (NmaLys), N-sigma-biotinyl-L-Lysine,N-sigma-9-fluorenylmethoxycarbonyl-L-Lysine, N-sigma-methyl-L-Lysine,N-sigma-dimethyl-L-Lysine, N-sigma-trimethyl-L-Lysine,N-sigma-isopropyl-L-Lysine, N-sigma-dansyl-L-Lysine,N-sigma-o,p-dinitrophenyl-L-Lysine, N-sigma-p-toluenesulfonyl-L-Lysine,N-sigma-DL-2-amino-2carboxyethyl-L-Lysine,N-sigma-phenylpyruvamide-L-Lysine, N-sigma-pyruvamide-L-Lysine.

For example, in a preferred method of manufacture as set out above thenon-natural amino acid contains an azide, alkyne, alkene, or aryl, alkylor vinyl halide, ketone, aldehyde, hydrazine, hydrazide, alkoxy amine,boronic acid, organotin, organosilicon group prior to linkage. Thenon-natural amino acid may be azidohomoalanine, homopropargylglycine,homoallylglycine, p-bromophenylalanine, p-iodophenylalanine,azidophenylalanine, acetylphenylalanine or ethynylephenylalanine, aminoacids containing an internal alkene such as trans-crotylalkene, serineallyl ether, allyl glycine, propargyl glycine, or vinyl glycine.

In a preferred method of manufacture as set out above the reaction forcoupling the first portion to the second portion is a [3+2] dipolarcycloaddition or Click reaction, a Heck reaction, a Sonogashirareaction, a Suzuki reaction, a Stille coupling, a Hiyama/Denmarkreaction, olefin metathesis, a Diels-alder reaction, or a carbonylcondensation with hydrazine, hydrazide, alkoxy amine or hydroxylamine.

PEGylation of Bivalent Bispecific Constructs and Antibodies

One of the drawbacks of recombinant bispecific antibodies known in theart is their short circulation time in the body. Diabodies, single-chaindiabodies and tandem-scFv molecules have a molecular weight of 50-60kDa., which can cause rapid clearance of these entities from thecirculation by extravasation, proteolysis and renal elimination.Exemplary initial half-lives of these entities (t1/2α) are below 30 min.Several approaches have been undertaken to improve the pharmacokineticsof recombinant antibodies. One approach is to increase the size of thesemolecules. Dimeric single-chain diabody molecules with a molecularweight of 100-115 kDa can also be generated by varying the length of thelinkers connecting the variable domain. Other approaches rely on theassociation of the bispecific to serum proteins that have longhalf-lives. These include the fusion of bispecific antibodies to humanserum albumin (HSA), HSA binding peptides, or to peptides derived fromhormones that have naturally long half-lives. Such methods may beapplied to the bivalent bispecific constructs of the present invention.However, the present invention also provides for the use ofpolyethylene-glycol polymers (PEG), which is shown for the first timeherein to be particularly advantageous in extending the half life of thebivalent bispecific constructs of the present invention.

PEG has several chemical properties which are desirable in a finalbispecific product and solve problems endemic with scFvs. PEGylationshould improve protein solubility and increase scFv stability, therebyreducing scFv aggregation and precipitation. In addition, a long andflexible linker such as PEG increases the physical separation of the twoantibody fragments, allowing them to refold independently from eachother. This solves one of the problems that often occurs in therefolding of bispecific antigen binding domains linked by geneticfusion, (i.e. uncontrolled and undesirable cross linking between the twoconstituent antibodies). PEG polymers are traditionally covalentlylinked to biomolecules through reactive sites such as lysine, cysteineand histidine residues. However, in order to achieve optimal stability,the amount of polymer attached to the target molecule needs to betightly controlled. Conjugation of PEG polymers to reactive sites in theprotein often results in a heterogeneous mixture of PEG-modifiedproteins, which may result in sub-optimal stabilization and half lifeextension, as well as potential loss of bioactivity of thepolymer-modified protein when the PEG reactive sites are important forthe protein activity (e.g. they are located in or near a receptorbinding site). The present invention provides a solution to thisproblem, by engineering the constituent antibodies of the bivalentbispecific construct to include non-natural amino acids at specificlocations and reacting PEG with these non-natural amino acids.

The use of a PEG linker provides yet more advantages to those detailedabove due to the versatility of the chemical syntheses that may be used.PEG can be easily functionalized to be a complementary reaction partnerwith any non-natural amino acid that is incorporated into the scFvproteins. PEG can also be functionalized with multiple sites ofconjugation which enables construction of multivalent protein hybrids.The PEG functionalization can be made with homo-bifunctional orhetero-bifunctional PEG's depending on the desired conjugationchemistry. In addition, the structure of PEG can be tailored for linearor branched variations, which can impact pharmacokinetics andbioactivity.

The preparation of these PEGylated bivalent bispecific constructs isdiscussed further below

Bispecific scFvs may be constructed by the conjugation of two differentscFv antigen binding domains to each other by way of a linker. Thisstrategy may be realized in a two-step process in which each scFv isconjugated to the bifunctional linker. The two scFvs, comprising thebispecific conjugate, each contain at least one non-natural amino acid(e.g. Aha) at a position which serves as a specific site of conjugation.The linker can be homo-bifunctional or hetero-bifunctional and contain acomplimentary functional group (e.g. alkyne) that is reactive with thenon-natural amino acid contained in the scFv (Aha). The followingreaction scheme can then be applied by to generate bispecific scFv(Scheme 1 below).

The chemistry used to conjugate scFvs to the linker is orthogonal to the20 natural amino acids. Azide-alkyne copper mediated cycloaddition isused here, in the preparation of scFv-PEG conjugates and bispecifics. Ina typical sequence, an scFv containing azidohomoalanine (Aha) is reactedwith an excess amount of a homo-bifunctional PEG linker functionalizedwith alkynes. The predominant product at limiting excess of PEG, is amonovalent PEGylated scFv, which is then purified. The free pendantalkyne of the PEG linker undergoes a second copper mediated azide-alkynecycloaddition with a second scFv containing Aha to afford thebispecific. Azide-alkyne copper mediated cycloadditions (Meldal andTornøe, 2008, Kolb et al 2001), as well as alkene-aryl halide palladiummediated Heck reactions, have been extensively applied to the sitespecific conjugation of polymers, toxins or peptides to target proteins.The copper mediated cycloaddition reaction is completely orthogonal withall natural amino acids, such that this chemistry cannot be used tomodify biological molecules, unless a non natural azide or alkynecontaining moiety can be introduced. When this is done, the chemistryoccurs only at the position of that azide or alkyne. Azides and alkynescan be introduced into proteins as analogs of natural amino acids,providing a specific position for bioconjugation.

As noted elsewhere, anti-IL-6 and anti-IL-23 or derivatives thereof,(including anti-IL-23/IL-12 antibodies, or derivatives thereof) mayoptionally be modified through PEGylation to increase half life.PEGylation of anti-IL-6 and anti-IL-23 or derivatives thereof may beachieved through similar methods.

Suitably PEG groups and PEG linkers of use in bispecific constructs andantibodies of the invention have weight 2-100 kDa for example 5-60 kDae.g. 10-40 kDa such as around 20 or around 40 kDa. PEG groups andlinkers may be straight chain or branched.

Pharmaceutical Compositions

In accordance with another aspect, the invention provides pharmaceuticalcompositions and kits comprising the bivalent, bispecific constructs ofthe invention and a pharmaceutically acceptable carrier. In someembodiments, the pharmaceutical composition or kit further comprisesanother component, such as an imaging reagent or therapeutic agent. Inpreferred embodiments, the pharmaceutical composition or kit is used indiagnostic or therapeutic methods.

Pharmaceutical compositions may for example be aqueous formulations e.g.aqueous solutions comprising conventional excipients such as sodiumchloride, sugars, amino acids, surfactants and the like.

Pharmaceutical compositions may also be lyophilized products suitablefor reconstitution by addition of water or saline.

Methods of Treatment

In accordance with another aspect, the invention provides for the use ofthe bivalent, bispecific constructs of the invention in therapy. Inparticular, the present invention provides for the treatment of T_(H)17,T_(H)22, and T_(H)1 mediated diseases, as well as diseases mediated bycombinations of these T_(H) cells.

Examples of such diseases that may be treated using the bivalent,bispecific constructs of the invention include inflammatory andautoimmune disorders, such as multiple sclerosis, psoriasis, psoriaticarthritis, pemphigus vulgaris, organ transplant rejection, Crohn'sdisease, inflammatory bowel disease (IBD), irritable bowel syndrome(IBS), lupus erythematosis, and diabetes.

Further examples of T_(H)17 mediated diseases include Amyotrophiclateral sclerosis or ALS (Lou Gehrig's disease), Ankylosing spondylitis,Asperger's, Back pain, Barrett's esophagus, Bipolar disorder, Cardiacarrhythmia, Celiac disease, Chronic fatigue syndrome (CFS/CFIDS/, E),Chronic Lyme disease (borreliosis), Crohn's disease, Diabetes insipidus,Diabetes type I, Diabetes type II, Dementia, Depression, Epilepsy,Fibromyalgia (FM), Gastroesophageal reflux disease (GERD), Hashimoto'sthyroiditis, Irritable Bowel Syndrome (IBS), Interstitial cystitis (IC),Inflammatory bowel disease, Irritable bowel syndrome, Kidney stones,Löfgren's syndrome, Lupus erythematosis, Mania, Multiple ChemicalSensitivity (MCS), Migraine headache, Morgellon's, Multiple sclerosis,Myasthenia gravis, Neuropathy, Obsessive Compulsive Disorder (OCD),Osteoarthritis, Panic attacks, Parkinson's, Polymyalgia rheumatic,Postural orthostatic , achycardia syndrome (POTS), Prostatitis,Psoriasis, Psoriatic arthritis, Raynaud's syndrome/phenomenon, Reactivearthritis (Reiter syndrome), Restless leg syndrome, Reflex SympatheticDystrophy (RSD), Rheumatoid arthritis, Sarcoidosis, Scleroderma,inusitis, Seasonal affective disorder (SAD), Sjögren's syndrome,Ulcerative colitis, Uveitis, and Vertigo. Further diseases includecytokine storm from sepsis or hemorrhagic fever, biliary cirrhosis,Still's Disease, COPD, Grave's Opthalmopathy, perionditis, BehcetDisease, asthma, atopic dermatitis, Hidradenitis suppurativa, Giant cellarteritis and cardiac fibrosis.

Further examples of T_(H)22 mediated diseases include chronicinflammatory diseases such as eczema, scleroderma, asthma and COPD.

Accordingly, in an embodiment the present invention provides a method oftreatment of T_(H)17 mediated diseases comprising administering atherapeutically effective amount of the bivalent bispecific construct ofthe present invention to a patient.

In another embodiment the present invention provides a bivalent,bispecific construct of the present invention for the treatment ofdiseases mediated by T_(H)17.

In another embodiment the present invention provides for the use of abivalent, bispecific construct of the present invention for themanufacture of a medicament for the treatment of diseases mediated byT_(H)17.

In another embodiment the present invention provides a method oftreatment of diseases mediated by T_(H)22 cells comprising administeringa therapeutically effective amount of the bivalent, bispecific constructof the present invention to a patient.

In another embodiment the present invention provides a bivalent,bispecific construct of the present invention for the treatment ofdiseases mediated by T_(H)22 cells.

In another embodiment the present invention provides for the use of abivalent, bispecific construct of the present invention for themanufacture of a medicament for the treatment of diseases mediated byboth T_(H)22 cells.

In another embodiment the present invention provides a method oftreatment of diseases mediated by both T_(H)17 and T_(H)1 cellscomprising administering a therapeutically effective amount of thebivalent, bispecific construct of the present invention to a patient

In another embodiment the present invention provides a bivalent,bispecific construct of the present invention for the treatment ofdiseases mediated by both T_(H)17 and T_(H)1.

In another embodiment the present invention provides for the use of abivalent, bispecific construct of the present invention for themanufacture of a medicament for the treatment of diseases mediated byboth T_(H)17 and T_(H)1.

In another embodiment the present invention provides a method oftreatment of T_(H)17 mediated diseases comprising administering atherapeutically effective amount of a combination of anti-IL-6 andanti-IL-23 antibodies or derivatives thereof according to the presentinvention to a patient.

In another embodiment the present invention provides a combination ofanti-IL-6 and anti-IL-23 antibodies or derivatives thereof according tothe present invention for the treatment of T_(H)17 mediated diseases.

In another embodiment the present invention provides for the use of acombination of anti-IL-6 and anti-IL-23 antibodies or derivativesthereof according to the present invention for the manufacture of amedicament for the treatment of T_(H)17 mediated diseases.

Accordingly, in an embodiment the present invention provides a method oftreatment of diseases mediated by T_(H)22 cells comprising administeringa therapeutically effective amount of a combination of anti-IL-6 andanti IL-23 antibodies or derivatives thereof according to the presentinvention to a patient.

In another embodiment the present invention provides a combination ofanti-IL-6 and anti-IL-23 antibodies or derivatives thereof according tothe present invention for the treatment of diseases mediated by T_(H)22cells.

In another embodiment the present invention provides for the use of acombination of anti-IL-6 and anti-IL-23 antibodies or derivativesthereof according to the present invention for the manufacture of amedicament for the treatment of diseases mediated by T_(H)22 cells.

Accordingly, in an embodiment the present invention provides a method oftreatment of diseases mediated by both T_(H)17 and T_(H)1 comprisingadministering a therapeutically effective amount of a combination ofanti-IL-6 and anti-IL-23 antibodies or derivatives thereof according tothe present invention to a patient.

In another embodiment the present invention provides a combination ofanti-IL-6 and anti-IL-23 antibodies or derivatives thereof according tothe present invention for the treatment of diseases mediated by bothT_(H)17 and T_(H)1.

In another embodiment the present invention provides for the use of acombination of anti-IL-6 and anti-IL-23 antibodies or derivativesthereof according to the present invention for the manufacture of amedicament for the treatment of diseases mediated by both T_(H)17 andT_(H)1.

The invention also provides methods of treatment of diseases that haveboth a T_(H)17 and T_(H)22 component to their aetiology. Additionally,the aetiology of the diseases to be treated according to the presentinvention may involve all three of T_(H)17, T_(H)22 and T_(H)1 cells.

In each of the embodiments listed above, the anti-IL-23 antibody, orderivative thereof may be an anti-II-23/IL-12 antibody

Dosage Regimes

In another aspect of the invention the bivalent bispecific constructs,antibodies, and antibody combinations for use in therapy according tothe present invention may be administered to patients at advantageouslylow doses whilst still achieving the same therapeutic effect , ascompared to therapies currently available in the art. The lower dosesare facilitated by higher activity of the antibodies disclosed herein,and potentially reduce the incidence of side effects.

Alternatively, if greater activity is desired the bivalent bispecificconstructs, antibodies, and antibody combinations for use in therapyaccording to the present invention may be administered to patients atequivalent or higher doses as compared to therapies currently availablein the art. Such higher dosages may facilitate reduced frequency ofadministering bivalent bispecific constructs, antibodies, and antibodycombinations to a patient

In an embodiment the bivalent bispecific constructs, antibodies, andantibody combinations of the present invention may be administeredmonthly, bi-monthly, weekly, bi-weekly, daily, bi-daily.

Assays for Determining IL-6, IL-23 and IL-23/IL-12 Antibody Affinitiesand Biological Activity Determination of Antibody Affinity

Antibody affinities may be determined using methods well known to theperson skilled in the art.

For the purposes of determining whether antibodies have the desiredaffinity to render them potentially suitable for inclusion in thebivalent bispecific antibodies of the present invention, the followingdetailed assay procedure is provided, but it will be appreciated thatminor variations to the methodology (e.g. in the use of different, butsimilar, pieces of apparatus, or different brands of common reagents)will allow for the same determination to be made.

Equilibrium dissociation constants may be determined by surface Plasmonresonance using a SensiQ Pioneer (ICx Nomadics, Stillwater, Okla.) and acarboxylated COOH1 sensor (Ibid) amenable for amine coupling.

Protein G (6510-10, Biovision, Mountain View, Calif.) is coupled to theCOOH1 sensor using amine coupling reagents (Sigma Aldrich(N-Hydroxysuccinimide (NHS, 56480),N-(3-Dimethylaminopropyl)-B′-ethylcarbodiimide hydrochloride (EDC,E7750), Ethanolamine (398136), St. Louis, Mo.) or with the Biacore AmineCoupling Kit (BR-1000-50, GE Healthcare, Waukesha, Wis.).

Briefly, the carboxylated surface is activated with 2 mM EDC and 0.5 mMNHS for a contact time ranging between 2-10 minutes. Protein G, invariable concentrations ranging between 20-400 ug/mL, is diluted into 10mM acetate buffer, pH 4.3 (sodium acetate, BP334-1; glacial acetic acid,A490-212; Thermo Fisher Scientific, Waltham, Mass.), and injected overthe activated sensor for variable contact times ranging between 5 and 10minutes at a rate ranging from 5-10 μL/min. Quantities of Protein Gimmobilized to the COOH1 sensor chip range from 400-2000 response units(RU). Remaining activated sites should be capped with 100 μL ofethanolamine at a flow rate of 25 μL/min.

Equilibrium constants for rabbit human chimeric mAbs may be determinedby binding the mAb to the protein G coated chip followed by binding ofeach analyte (IL-6 or IL-23) to its respective mAb. In order to minimizemass transfer effects, the surface densities of the mAbs for eachanalyte should be adjusted so that as analyte binding approachessaturation its corresponding RU falls between 200 and 300. Dilutions of3×-FLAG-IL-6 (see example 1), IL-6 (CYT-213, Prospec-Tany Technogene,Rehovot, Israel) or human dimeric IL-23 (34-8239, eBiosciences, SanDiego, Calif.) ranging from 1 to 100 nM are injected over the chipsurface and association (Ka) and dissociation (Kd) rate constants aremeasured. For each binding and dissociation cycle the chip surfaceshould be regenerated with 15 uL of 20 mM NaOH (5671-02, MallinckrodtBaker, Philliphsburg, N.J.). The assay temperature should be maintainedat 25° C. with an analyte flow rate of 50 μL/min and include a 2 minuteassociation phase and 10-30 minute dissociation phase. The on/off rates(ka/kd) and dissociation constants (KD) may be determined using theformat described above along with pseudo-first-order 1:1 interactionmodel software (Qdat, ICx Nomadics, Stillwater, Okla.).

Equilibrium constants for scFvs may be determined as previouslydescribed; with the proviso that the protocol should be modified so thatan epitope tagged IL-6 is captured on the chip surface and thedissociation of anti-IL-6 scFvs from IL-6 is monitored. Briefly,anti-FLAG® M2 antibody (200472, Agilent Technologies, Santa Clara,Calif.) is bound to Protein G, and then 3×FLAG-IL-6 is captured by theanti-FLAG antibody. The anti-IL-6 scFvs should be assayed over a rangeof concentrations between 1 and 100 nM.

SPR of Bispecific scFvs

SPR of bispecific scFvs are carried out as previously described formeasuring the anti-IL-6 moiety; with the proviso that the protocol ismodified in order to also determine the binding kinetics of the attachedanti-IL-23 scFv, 31A12, at the other end of the bispecific. Briefly,IL-23 binding by the bispecific was performed by first immobilizing thebispecific with IL-6 as described, at a constant density (˜240 RU).Binding and dissociation of a recombinant human dimeric IL-23 (34-8239,eBiosciences, San Diego, Calif.) may be assayed in concentrationsranging from 3 to 25 nM using the same parameters detailed above

Determination of IL-6 Activity

The ability of the antibodies and derivatives thereof to modulate IL-6activity may be assayed using methods well known to the person skilledin the art. For the purposes of determining whether antibodies have thedesired ability to modulate IL-6 activity that would render thempotentially suitable for inclusion in the bivalent bispecific antibodiesof the present invention, the following detailed assay procedure isprovided, but it will be appreciated that minor variations to themethodology (e.g. in the use of different, but similar, pieces ofapparatus, or different brands of common reagents) will allow for thesame determination to be made.

An ELISA may be used to evaluate IL-6 binding. Recombinant IL-6 (SeeExample 1) is added to an ELISA plate in 100 μl PBS at 0.25 μg/ml.Plates should be incubated 1 hour at 37C, or overnight at 4C. To block100 μl/well PBS containing 10% goat serum (Cat #16210-072, Invitrogen,USA) should be added to each well. Plates should then be incubated 1hour at room temperature. Plates should then be rinsed 5 times withde-ionized water. To each well is added 50 μl PBS/10% goat serum. Testsamples are then added at 50 μl/well. Plates should then be incubated 1hour at room temperature. Plates should then be rinsed 5 times withde-ionized water. To each well is added 100 μl peroxidase-conjugatedgoat anti-rabbit IgG (Cat. #111-035-008, Jackson Immuno Research)diluted 1:5000 in PBS/10% goat serum. Plates should then be incubated 1hour at room temperature, then washed 5 times with de-ionized water. TMBsubstrate (Thermo Scientific, Rockford, Ill., USA) is added at 100μl/well. The reaction should then be stopped with 100 μl 1N H2SO4 (JTBaker, Phillipsburg, N.J., USA). Absorbance can then be measured at 450nm using a Molecular Devices M2 plate reader.

A bioassay using an IL-6 dependant murine B-cell hybridoma cell line(B9cell line; Aarden et al., 1987) may be used to evaluate IL-6inhibition (FIG. 3). Samples to be tested for neutralizing activityshould be diluted in 100 μl assay medium (RPMI 1640 w/L-glutamine, 10%FBS, Non-Essential Amino Acids, Sodium Pyruvate, 50 μM2-mercaptoethanol) in a 96-well tissue culture plate. This is followedby the addition of 50 μl of IL-6 (Cat. # CYT-274 Prospec-TanyTechnogene) containing assay medium, and 30 minutes of incubation atroom temperature. B9 cells are then recovered from flasks andcentrifuged for 7 min at 180×g, and the pellet resuspended in IL-6-freeculture medium (RPMI 1640 w/L-glutamine, 10% FBS, Non-Essential AminoAcids, Sodium Pyruvate, 50 μM 2-mercaptoethanol). Cells should becentrifuged and resuspended three times to remove IL-6. Followingviability determination by trypan blue exclusion, cells should beadjusted to 1×105 cells/ml. A volume of 50 μl of B9 cells, correspondingto 5×10³ cells, should be added to each well along with appropriatecontrol wells containing IL-6-free medium.

The plates should then be incubated for 48 h at 37° C. 5% CO2.Subsequently, 20 μl of Alamar Blue (Cat # DAL1100, Invitrogen, USA)should be added to each well, and the plates incubated for an additional18 h. The plates can then be read on a Molecular Devices (Sunnyvale,Calif., USA) M2 plate reader at 570 and 600 nm.

Determination of IL-23 Activity

An ELISA assay may be used to evaluate IL-23 binding (Aggarwal et al.,2003). ELISA plates are coated using either a direct or indirect methodof binding IL-23.

For the indirect binding method anti-His antibody (Cat # A00613,GenScript Corp., New Jersey, USA) should be added to the plates in 100ml/well of PBS at 0.01-0.02 ug/ml. Plates should then be incubated for 1hour at 37C, or overnight at 4C. To block non-specific binding 100ml/well PBS containing 10% goat serum (Cat #16210-072, Invitrogen, USA)should be added to each well, after which plates should be rinsed 5times with de-ionized water. IL-23 p40-p19-His (SEQ ID 4) in 100 ml/wellPBS/10% goat serum at 0.5 mg/ml should be added and the plates incubatedfor 1 hour at room temperature.

For the direct binding method IL-23 p40-p19-His (SEQ ID NO. 4) should beadded to an ELISA plate in 100 ml PBS at 0.5 mg/ml. Plates should thenbe incubated for 1 hour at 37C, or overnight at 4C. To blocknon-specific binding 100 ml/well PBS containing 10% goat serum (Cat#16210-072, Invitrogen, USA) should be added to each well. Plates shouldthen be incubated for 1 hour at room temperature.

After IL-23 binding, plates should be rinsed 5 times with de-ionizedwater. To each well should be added 50 ml PBS/10% goat serum. Testsamples should then be added at 50 ml/well. Plates should then beincubated for 1 hour at room temperature and rinsed 5 times withde-ionized water. To each well should then be added 100 mlperoxidase-conjugated goat anti-rabbit IgG (Cat. #111-035-008, JacksonImmuno Research) diluted 1:5000 in PBS/10% goat serum. Plates shouldthen be incubated 1 hour at room temperature, then washed 5 times withde-ionized water. TMB substrate (Thermo Scientific, Rockford, Ill., USA)should be added at 100 ml /well. The reaction should then be stoppedwith 100 ml 1N H2SO4 (JT Baker, Phillipsburg, N.J., USA). Absorbance canthen be measured at 450 nm using a Molecular Devices M2 plate reader.

A bioassay, based on the detection of IL-23-induced IL-17 expression bymouse spleen cells, may be used to detect antibody mediated inhibitionof IL-23 binding to the IL-23 receptor and resulting bioactivity.

5×10⁵ C57Bl/6 spleen cells should be cultured in the wells of a 96-wellplate in 200 ml containing a dilution of the heterodimeric IL-23(eBioscience cat. #14-8239 or Humanzyme, Chicago, USA cat. #HZ-1049) andthe plates incubated for 2-3 days at 37° C. The culture medium usedshould be RPMI 1640, 10% FBS, 50 uM 2-mercaptoethanol, non-EssentialAmino Acids, pyruvate, gentamicin and 10 ng/ml human IL-2 (Cat #CYT-209, Prospec-Tany Technogene). After 3 days, the culturesupernatants should be assayed by ELISA for IL-17A, as described below.

An ELISA assay may be used used to detect mouse IL-17. Plates are coatedwith anti-mlL-17A (eBioscience #14-7178) 1 mg/ml in 100 ml PBS, andincubated overnight at 4° C. or 1 hr at 37° C. Plates should be washedin deionized water and blocked for 1 h with 100 ml of PBS, 10% goatserum. After washing the plates, 50 ml of PBS/10% goat serum and 50 mlof culture supernatant should be added to the plates, and incubated for1 hr. The plates should then be washed, 100 ml/well ofanti-mlL-17A-Biotin (eBioscience #13-7179) at 0.5 mg/ml in PBS/10% goatserum added and the plates incubated for 1 h at room temperature. Theplates should then be washed, and reacted with 100 ml/wellStreptavidin-HRP (Jackson Labs) at 1:1000 in PBS/10% goat serum. Platesshould be washed again, and the signal detected by adding 100 ml/wellTMB substrate (Thermo Scientific, IL, USA). After stopping the reactionwith 100 ml/well 1N H2SO4, the optical density can be read at 450 nM.

Determination of IL-12 (p40) Activity

In addition, given that the p40 subunit of IL-23 also forms part ofIL-12, which is involved in the T_(H)1 signaling pathway, an assay tomeasure their neutralizing capacity against IL-12 utilizes their abilityto modulate the level of IFN-γ (a product of T_(H)1 cell activity) aredisclosed herein. The person skilled in the art will be aware ofsuitable assay methods to determine the neutralizing effect of theantibodies and their effect on IFN-γ production, however, the followingassay is provided as an example of a suitable assay.

Antibodies may be assayed for p40 neutralizing capacity using the IL-12responsive cell line NK-92 (CRL-2407, ATCC, Manassas, Va., USA). 50 mlof culture supernatant from the B cell cloning plates or 50 ml ofsupernatant from antibody transfection should be transferred to a 96well tissue culture plate. 50 ml of human IL-12 (Cat. # Cyt-362,Prospec-Tany Technogene, Rehovot, Israel) should be added to each wellat 4 ng/ml. Plates should then be incubated for 30-60 minutes at roomtemperature, after which 5×10⁴ NK-92 cells should be added to each wellin 100 ml. Cultures should then be incubated for 3 days at 37° C., andtheir supernatants assayed for human Interferon-γ production. The assaymedium should be RPMI 1640, 10% FBS, NEAA, pyruvate, 50 mM2-mercaptoethanol, gentamicin and 10 ng/ml human IL-2 (Cat # Z00368,GeneScript Corporation, Piscataway, N.J., USA).

An ELISA assay may be used to detect human Interferon-γ. Plates arecoated with anti-human Interferon-g (Cat. # Mab 1-D1K, Mabtech,Cincinnati, Ohio, USA) 1 mg/ml in 100 ml PBS, overnight @4° C. or 1 hr @37° C. Plates should then be washed in de-ionized water and blocked for1 h with 100 μl of PBS, 10% goat serum. After washing the plates, 50 mlof PBS/10% goat serum and 50 ml of culture supernatant were added to theplates, and incubated for 1 hr. The plates should then be washed, 100ml/well of anti-human Interferon-γ-Biotin (Cat # Mab 7b6-1-biotin,Mabtech) at 0.5 mg/ml in PBS/10% goat serum added and the plates thenincubated for 1 h at room temperature. The plates should then be washedand reacted with 100 ml/well Streptavidin-HRP (Jackson Labs) at 1:1000in PBS/10% goat serum. Plates should then be washed again, and thesignal detected by adding 100 ml/well TMB substrate (Thermo Scientific,IL, USA). After stopping the reaction with 100 ml/well 1N H2SO4, theoptical density can be read at 450 nM.

Reactivity Against Primate Interleukins (IL-6, IL-23 and IL-12)

The assays against primate interleukins are identical to those for themeasurement of activity against the human assays, save for the use ofthe primate version of the cytokine being assayed.

The successful development of any cytokine antagonist for human therapywill require initial toxicology testing. Toxicology is most efficientlydemonstrated in non human species. In order to facilitate initialtoxicology studies the antibodies of the present invention may bescreened for their ability to neutralize IL-6 from the species beingconsidered for the studies.

DEFINITIONS

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

An “immunoglobulin” is a tetrameric molecule. In a naturally-occurringimmunoglobulin, each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one“light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The amino-terminal portion of eachchain includes a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The carboxy-terminalportion of each chain defines a constant region primarily responsiblefor effector function. Human light chains are classified as K and Xlight chains. Heavy chains are classified as, a, or E, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. See generally,Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)) (incorporated by reference in its entirety for all purposes).The variable regions of each light/heavy chain pair form the antibodybinding site such that an intact immunoglobulin has two binding sites.

Immunoglobulin chains exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hypervariable regions,also called complementarity determining regions or CDRs. The CDRs fromthe two chains of each pair are aligned by the framework regions,enabling binding to a specific epitope. From N-terminus to C-terminus,both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4. The assignment of amino acids to each domain is inaccordance with the definitions of Kabat Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.(1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196: 901-917 (1987);Chothia et al. Nature 342: 878-883 (1989).

An “antibody” refers to an intact immunoglobulin, or to anantigen-binding portion thereof that competes with the intact antibodyfor specific binding. Antigen-binding portions may be produced byrecombinant DNA techniques or by enzymatic or chemical cleavage ofintact antibodies. Antigen-binding portions include, inter alia, Fab,Fab′, F (ab′)2, Fv, dAb, and complementarity determining region (CDR)fragments, single-chain antibodies (scFv), chimeric antibodies,diabodies and polypeptides that contain at least a portion of animmunoglobulin that is sufficient to confer specific antigen binding tothe polypeptide. An Fab fragment is a monovalent fragment consisting ofthe VL, VH, CL and CH I domains; a F (ab′) 2 fragment is a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; a Fd fragment consists of the VH and CH 1 domains; anFv fragment consists of the VL and VH domains of a single arm of anantibody; and a dAb fragment (Ward et al., Nature 341: 544-546, 1989)consists of a VH domain. A single-chain antibody (scFv) is an antibodyin which a VL and VH regions are paired to form a monovalent moleculesvia a synthetic linker that enables them to be made as a single proteinchain (Bird et al., Science 242: 423-426, 1988 and Huston et al., Proc.Natl. Acad. Sci. USA 85: 5879-5883, 1988).

An antibody may have one or more binding sites. If there is more thanone binding site, the binding sites may be identical to one another ormay be different. For instance, a naturally-occurring immunoglobulin hastwo identical binding sites, a single-chain antibody or Fab fragment hasone binding site, while a “bispecific” antibody has two differentbinding sites. Bispecific antibodies can be produced by a variety ofmethods including fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321(1990), Kostelny et al. J. Immunol. 148: 1547-1553 (1992).

An “isolated antibody” is an antibody that (1) is not associated withnaturally-associated components, including other naturally-associatedantibodies, that accompany it in its native state, (2) is free of otherproteins from the same species, (3) is expressed by a cell from adifferent species, or (4) does not occur in nature. Examples of isolatedantibodies include an anti-IL-6 antibody that has been affinity purifiedusing IL-6, an anti-IL-6 antibody that has been synthesized by ahybridoma or other cell line in vitro, and a human anti-IL-6 antibodyderived from a transgenic mouse.

The term “human antibody” includes all antibodies that have one or morevariable and/or constant regions derived from human immunoglobulinsequences. These antibodies may be prepared in a variety of ways, by wayof example two are described below.

A humanized antibody is an antibody that is derived from a non-humanspecies, in which certain amino acids in the framework and constantdomains of the heavy and light chains have been mutated so as to avoidor abrogate an immune response in humans.

Alternatively, a humanized antibody may be produced by fusing theconstant domains from a human antibody to the variable domains of anon-human species. Examples of how to make humanized antibodies may befound in U.S. Pat. Nos. 6,054,297, 5,886,152 and 5,877,293.

The term “chimeric antibody” refers to an antibody that contains one ormore regions from one antibody and one or more regions from one or moreother antibodies.

The term “Koff” refers to the off rate constant for dissociation of anantibody from the antibody/antigen complex.

The term “Kd” refers to the dissociation constant of a particularantibody-antigen interaction.

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor. Epitopic determinantsusually consist of chemically active surface groupings of molecules suchas amino acids or sugar side chains and usually have specific threedimensional structural characteristics, as well as specific chargecharacteristics. An antibody is said to specifically bind an antigenwhen the dissociation constant is, preferably less than 10 nM and mostpreferably 0 nM.

Fragments or analogs of antibodies or immunoglobulin molecules can bereadily prepared by those of ordinary skill in the art following theteachings of this specification.

Preferred amino- and carboxy-termini of fragments or analogs occur nearboundaries of functional domains. Structural and functional domains canbe identified by comparison of the nucleotide and/or amino acid sequencedata to public or proprietary sequence databases.

“IL-12” is a heterodimer consisting of two subunits, p35 and p40, linkedby a disulfide bond. Antigen presenting cells, primarily of the myeloidlineage, express IL-12, which participates in cell-mediated immunity bybinding to a receptor complex expressed on the surface of T cells ornatural killer cells. It is believed that the p40 subunit of IL-12 bindsthe IL-12 receptor beta 1 (IL-12Rβ1) receptor and the p35 subunit bindsto the second receptor chain (IL-12Rβ2), resulting in intracellularsignaling.

“IL-23” is a heterodimer, consisting of the same p40 protein subunit ofIL-12, covalently linked to a p19 protein. IL-23 binds to a receptorrelated to the IL-12R, that shares the IL-12Rβ1 chain and also has aunique IL-23R chain.

“IL-6” is a pleiotropic cytokine with various biological activities inimmune regulation including hematopoiesis, inflammation, andoncogenesis. IL-6 activates a receptor complex consisting of the IL-6receptor (IL-6R) and the signal-transducing receptor subunit gp130.IL-6R exists in both a transmembrane form and a soluble form. IL-6 bindsto both of these forms, which can then interact with gp130 to triggerdownstream signal transduction and gene expression.

“T_(H)1 cells” are T regulatory cells (also known as T helper cells)involved in mammalian immune responses. They are characterized by theproduction of IFN-γ.

“T_(H)17 cells” are T regulatory cells (also known as T helper cells)involved in mammalian immune responses. They are characterized by theproduction of IL-17.

“T_(H)17 mediated diseases” are diseases in which T_(H)17 cells play arole in the aetiology of the disease.

“T_(H)22 cells” are T regulatory cells (also known as T helper cells)involved in mammalian immune responses. They are characterized by theproduction of IL-22.

“T_(H)22 mediated diseases” are diseases in which T_(H)22 cells play arole in the aetiology of the disease.

Preferably, computerized comparison methods are used to identifysequence motifs or predicted protein conformation domains that occur inother proteins of known structure and/or function. Methods to identifyprotein sequences that fold into a known three-dimensional structure areknown. Bowie et al. Science 253: 164 (1991).

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain (s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et al. Nature 354: 105 (1991), which are each incorporatedherein by reference.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology-A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), which is incorporated herein by reference.Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as a-,a-disubstituted amino acids,N-alkyl amino acids, lactic acid, and other unconventional amino acidsmay also be suitable components for polypeptides of the presentinvention. Examples of unconventional amino acids include:4-hydroxyproline, -carboxyglutamate, s-N,N,N-trimethyllysine,s-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, s-N-methylarginine, and othersimilar amino acids and imino acids (e.g., 4-hydroxyproline). In thepolypeptide notation used herein, the lefthand direction is the aminoterminal direction and the righthand direction is the carboxy-terminaldirection, in accordance with standard usage and convention.

The term“polynucleotide” as referred to herein means a polymeric form ofnucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes single and double stranded forms of DNA.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the“isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in whichthe“isolated polynucleotide” is found in nature, (2) is operably linkedto a polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence.

The term “oligonucleotide” referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. Preferably oligonucleotides are 10 to 60bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or20 to 40 bases in length. Oligonucleotides are usually single stranded,e.g. for probes; although oligonucleotides may be double stranded, e.g.for use in the construction of a gene mutant. Oligonucleotides of theinvention can be either sense or antisense oligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term“modified nucleotides”referred to herein includes nucleotides with modified or substitutedsugar groups and the like. The term “oligonucleotide linkages” referredto herein includes oligonucleotides linkages such as phosphorothioate,phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the like.See e.g., LaPlanche et al. Nucl. Acids Res. 14: 9081 (1986); Stec et al.J. Am. Chem. Soc. 106: 6077 (1984); Stein et al. Nucl. Acids Res. 16:3209 (1988); Zon et al. Anti-Cancer Drug Design 6: 539 (1991); Zon etal. Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F.Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec etal. U.S. Pat. No. 5,151,510; Uhlmann and Peyman Chemical Reviews 90: 543(1990), the disclosures of which are hereby incorporated by reference.An oligonucleotide can include a label for detection, if desired.

Unless specified otherwise, the left hand end of single-strandedpolynucleotide sequences is the 5′ end; the left hand direction ofdouble-stranded polynucleotide sequences is referred to as the 5′direction. The direction of 5′ to 3′ addition of nascent RNA transcriptsis referred to as the transcription direction; sequence regions on theDNA strand having the same sequence as the RNA and which are 5′ to the5′ end of the RNA transcript are referred to as “upstream sequences”;sequence regions on the DNA strand having the same sequence as the RNAand which are 3′ to the 3′ end of the RNA transcript are referred toas“downstream sequences”.

“Operably linked” sequences include both expression control sequencesthat are contiguous with the gene of interest and expression controlsequences that act in trans or at a distance to control the gene ofinterest. The term “expression control sequence” as used herein refersto polynucleotide sequences which are necessary to effect the expressionand processing of coding sequences to which they are ligated. Expressioncontrol sequences include appropriate transcription initiation,termination, promoter and enhancer sequences; efficient RNA processingsignals such as splicing and polyadenylation signals; sequences thatstabilize cytoplasmic mRNA ; sequences that enhance translationefficiency (i.e., Kozak consensus sequence); sequences that enhanceprotein stability; and when desired, sequences that enhance proteinsecretion. The nature of such control sequences differs depending uponthe host organism; in prokaryotes, such control sequences generallyinclude promoter, ribosomal binding site, and transcription terminationsequence; in eukaryotes, generally, such control sequences includepromoters and transcription termination sequence. The term “controlsequences” is intended to include, at a minimum, all components whosepresence is essential for expression and processing, and can alsoinclude additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences.

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome.

Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “expressionvectors”). In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

The term “selectively hybridize” referred to herein means to detectablyand specifically bind. Polynucleotides, oligonucleotides and fragmentsthereof in accordance with the invention selectively hybridize tonucleic acid strands under hybridization and wash conditions thatminimize appreciable amounts of detectable binding to nonspecificnucleic acids. “High stringency” or “highly stringent” conditions can beused to achieve selective hybridization conditions as known in the artand discussed herein. An example of “high stringency” or “highlystringent” conditions is a method of incubating a polynucleotide withanother polynucleotide, wherein one polynucleotide may be affixed to asolid surface such as a membrane, in a hybridization buffer of 6×SSPE orSSC, 50% formamide, 5×Denhardt's reagent, 0.5% SDS, 100 p, g/mldenatured, fragmented salmon sperm DNA at a hybridization temperature of42 C for 12-16 hours, followed by twice washing at 55 C using a washbuffer of 1×SSC, 0.5% SDS. See also Sambrook et al., supra, pp.9.50-9.55.

Two amino acid sequences are homologous if there is a partial orcomplete identity between their sequences. For example, 85% homologymeans that 85% of the amino acids are identical when the two sequencesare aligned for maximum matching. Gaps (in either of the two sequencesbeing matched) are allowed in maximizing matching; gap lengths of 5 orless are preferred with 2 or less being more preferred. Alternativelyand preferably, two protein sequences (or polypeptide sequences derivedfrom them of at least 30 amino acids in length) are homologous, as thisterm is used herein, if they have an alignment score of at more than 5(in standard deviation units) using the program ALIGN with the mutationdata matrix and a gap penalty of 6 or greater. See Dayhoff, M. O., inAtlas of Protein Sequence and Structure, pp. 101-110 (Volume 5, NationalBiomedical Research Foundation (1972)) and Supplement 2 to this volume,pp. 1-10. The two sequences or parts thereof are more preferablyhomologous if their amino acids are greater than or equal to 50%identical when optimally aligned using the ALIGN program.

The term “corresponds to” is used herein to mean that a polynucleotidesequence is identical to all or a portion of a reference polynucleotidesequence, or that a polypeptide sequence is identical to a referencepolypeptide sequence. In contrast, the term “complementary to” is usedherein to mean that the complementary sequence is identical to all or aportion of a reference polynucleotide sequence. For illustration, thenucleotide sequence “TATAC” corresponds to a reference sequence “TATAC”and is complementary to a reference sequence“GTATA”.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotide or amino acid sequences: “referencesequence”, “comparison window”, “sequence identity”, “percentage ofsequence identity”, and“substantial identity”. A “reference sequence” isa defined sequence used as a basis for a sequence comparison; areference sequence may be a subset of a larger sequence, for example, asa segment of a full-length cDNA or gene sequence given in a sequencelisting or may comprise a complete cDNA or gene sequence. Generally, areference sequence is at least 18 nucleotides or 6 amino acids inlength, frequently at least 24 nucleotides or 8 amino acids in length,and often at least 48 nucleotides or 16 amino acids in length. Since twopolynucleotides or amino acid sequences may each (1) comprise a sequence(i.e., a portion of the complete polynucleotide or amino acid sequence)that is similar between the two molecules, and (2) may further comprisea sequence that is divergent between the two polynucleotides or aminoacid sequences, sequence comparisons between two (or more) molecules aretypically performed by comparing sequences of the two molecules over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window”, as used herein, refers to aconceptual segment of at least 18 contiguous nucleotide positions or 6amino acids wherein a polynucleotide sequence or amino acid sequence maybe compared to a reference sequence of at least 18 contiguousnucleotides or 6 amino acid sequences and wherein the portion of thepolynucleotide sequence in the comparison window may comprise additions,deletions, substitutions, and the like (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch J. Mol. Biol. 48: 443 (1970), by the search forsimilarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S. A.)85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison,Wis.), Geneworks, or MacVector software packages), or by inspection, andthe best alignment (i.e. resulting in the highest percentage of homologyover the comparison window) generated by the various methods isselected.

The term “sequence identity” means that two polynucleotide or amino acidsequences are identical (i.e., on a nucleotide-by-nucleotide orresidue-by-residue basis) over the comparison window. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) or residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the comparison window (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide or amino acid sequence,wherein the polynucleotide or amino acid comprises a sequence that atleast 90 to 95 percent sequence identity, more preferably at least 98percent sequence identity, more usually at least 99 percent sequenceidentity as compared to a reference sequence over a comparison window ofat least 18 nucleotide (6 amino acid) positions, frequently over awindow of at least 24-48 nucleotide (8-16 amino acid) positions, whereinthe percentage of sequence identity is calculated by comparing thereference sequence to the sequence which may include deletions oradditions which total 20 percent or less of the reference sequence overthe comparison window. The reference sequence may be a subset of alarger sequence.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity, even morepreferably at least 98 percent sequence identity and most preferably atleast 99 percent sequence identity.

Preferably, residue positions which are not identical differ byconservative amino acid substitutions. Conservative amino acidsubstitutions refer to the interchangeability of residues having similarside chains. For example, a group of amino acids having aliphatic sidechains is glycine, alanine, valine, leucine, and isoleucine; a group ofamino acids having aliphatic-hydroxyl side chains is serine andthreonine; a group of amino acids having amide-containing side chains isasparagine and glutamine; a group of amino acids having aromatic sidechains is phenylalanine, tyrosine, and tryptophan; a group of aminoacids having basic side chains is lysine, arginine, and histidine; and agroup of amino acids having sulfur-containing side chains is cysteineand methionine. Preferred conservative amino acids substitution groupsare: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid sequence maintain at least 90%, more preferably 95%, andmost preferably 99% sequence identity. In particular, conservative aminoacid replacements are contemplated. Conservative replacements are thosethat take place within a family of amino acids that are related in theirside chains. Genetically encoded amino acids are generally divided intofamilies: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine; (3) non-polar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. Morepreferred families are: serine and threonine are aliphatic-hydroxyfamily; asparagine and glutamine are an amide-containing family;alanine, valine, leucine and isoleucine are an aliphatic family; andphenylalanine, tryptophan, and tyrosine are an aromatic family. Forexample, it is reasonable to expect that an isolated replacement of aleucine with an isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar replacement of an amino acid witha structurally related amino acid will not have a major effect on thebinding or properties of the resulting molecule, especially if thereplacement does not involve an amino acid within a framework site.Whether an amino acid change results in a functional peptide can readilybe determined by assaying the specific activity of the polypeptidederivative. Assays are described in detail herein.

As used herein, the terms “label” or “labelled” refers to incorporationof another molecule in the antibody. In one embodiment, the label is adetectable marker, e.g., incorporation of a radiolabelled amino acid orattachment to a polypeptide of biotinyl moieties that can be detected bymarked avidin (e.g., streptavidin containing a fluorescent marker orenzymatic activity that can be detected by optical or colorimetricmethods). In another embodiment, the label or marker can be therapeutic,e.g. a drug conjugate or toxin. Various methods of labeling polypeptidesand glycoproteins are known in the art and may be used.

Examples of labels include, but are not limited to, the following:radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc,111In, 125I, 131I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,-galactosidase, luciferase, alkaline phosphatase), chemiluminescentmarkers, biotinyl groups, predetermined polypeptide epitopes recognizedby a secondary reporter (e.g., leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags),magnetic agents, such as gadolinium chelates, toxins such as pertussistoxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. In some embodiments, labels are attached by spacerarms of various lengths to reduce potential steric hindrance.

The term “patient” includes human and veterinary subjects.

Throughout this specification and claims, the word “comprise” orvariations such as “comprises” or “comprising” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

Examples of the Invention

Antigen binding domains are generated from hyperimmunized rabbits fromcloned antigen specific B cells. Antigen specific B cells are selectedfrom rabbit blood by antigen panning and cloned under culture conditionsthat favor the expansion of activated B cells.

Example 1 Construction of IL6 and IL23 Expression Clones andPurification of Fusion Proteins

Human IL-6 (DQ891463; ABM82389.1, SEQ ID NOs. 343 and 344) was expressedas a 3×FLAG-IL6-Avi fusion. The expression construct was generated by atwo-step polymerase chain reaction (PCR) amplification. The product wasinserted into the plasmid p3xFLAG-CMV-23 (Sigma), downstream of the CMVpromoter and in frame with the pre-pro-trypsin leader sequence andtriple FLAG tag. The expressed IL-6 (SEQ ID NO. 1) contained an aminoterminal triple FLAG tag (amino acid sequence dhdgdykdhdidykdddd; SEQ IDNO. 345) and a carboxyl-terminal Avi tag (glndifeaqkiewhe; SEQ IS NO.346) (ALLO66-MM4-80) The IL6 coding region was amplified by PCR andinserted into p3xFLAG-CMV-23 to express 3×FLAG-IL6 and 3×FLAG-IL6-mycfusions. (SEQ ID NO. 2)

The coding region of the macaca mulatta IL-6 (mmIL-6; NM_(—)001042733.1;NP_(—)001036198; SEQ ID NO. 346 and SEQ ID NO. 347) was generated byoverlapping oligomers and PCR (DNA2.0). The synthesized sequence encodesmmIL-6 containing an amino terminal signal sequence(mnsfstsafgpvafslglllylpaafpap; SEQ IS NO. 349) and a carboxyl terminalFLAG tag. This construct was inserted into the mammalian expressionvector pCEP4 (Invitrogen) downstream of the CMV promoter(ALLO66-MM4-143). (SEQ ID NO. 3)

The human IL-23 cytokine is a functional heterodimer of the p19 (IL23A;accession number NT_(—)029419) and p40 (IL12B; NT 023133) proteins. TheIL-23 dimer was expressed as a p40:p19 fusion with the individualproteins separated by the elastin linker (accession numberNM_(—)001081755; SEQ ID NO. 351) gttcctggagtaggggtacctggggtgggc encodingthe amino acid sequence VPGVGVPGVG SEQ ID NO. 352). A two step PCRamplification strategy was used to generate a p40:p19 genetic fusion andalso introduce a 6×HIS tag on each of the domains. The resultingconstructs encoding p40:p19-6×HIS (SEQ ID NO. 4) and p40-6×HIS: p19 wereintroduced into the mammalian expression plasmid pCEP4 (Invitrogen)

Primate IL23 heterodimer was expressed as a p40 (NP_(—)001038190;NM_(—)001044725):p19 (BV209310) fusion with the elastin linker betweenthe two proteins and containing a 3′ 6×HIS tag. The construct wassynthesized by overlapping oligomers and PCR and cloned into thep3xFLAG-CMV-13 plasmid, downstream of the CMV promoter and in frame withthe Pre-pro-trypsin signal sequence (ALLO87-MM-5-74) (SEQ ID NO. 5).

Mammalian expression and purification of target proteins:

The expression of mammalian cytokines was performed in HEK293 orHEK293c18 cells. Transfections were performed using 3 μl/mg DNA oflipofectamine2000, or 293fectin (Invitrogen) on cells plated at adensity of approx. 200,000 cells/cm², and using 2-2.5 μg DNA per millioncells. Cells were incubated at 37° C. for 3-4 days in DMEM containing10% fetal calf serum and the growth medium collected for purification ofthe target protein. For the purification 6×HIS tagged proteins 1/10volume of 10× binding buffer (500 mM phosphate buffer pH7.5, 3M NaCl,200 mM imidazole, 1% Tween-20) was added to the culture medium anddialysed overnight at 4 C against PBS. Ni-NTA beads were added to theextracts for 2-3 hrs at 4° C. with end-over-end mixing. Beads werecollected by centrifugation and washed in Ni-NTA wash buffer (50 mMPhosphate, 300 mM NaCl, 20 mM imidazole, 0.1% Tween). Proteins bound tothe Ni-NTA beads were eluted by elution buffer (50 mM Phosphate bufferpH7.5, 300 mM NaCl, 500 mM imidazole). Fractions containing the targetprotein were identified by SDS-PAGE and Coomassie staining andquantified by densitometry. Peak fractions were combined and dialysed toPBS and used directly in in vitro assays or SPR analyses.

FLAG tagged proteins were purified from the expression medium usingM2-conjugated beads (Invitrogen). In short, M2 beads were added directlyto the expression medium and incubated at 4° C. for 4-16 hours. Thebeads were washed in FLAG wash buffer (20 mM Tris, pH7.4, 150 mM NaCl,0.1% Tween-20, 1 mM ethylenediaminetetraacetic acid) and bound proteincollected by washes with 0.1M glycine (pH2.5) or using 3×FLAG peptide.Acid elutions were immediately neutralized using 1/20^(th) volume of 1 MTris base. Peak fractions were identified by SDS-PAGE and Coomassiestaining and pooled. These were dialysed to PBS and used directly for invitro assays or SPR analyses.

Example 2 Determination of Antibody Affinity

Equilibrium dissociation constants were determined by surface Plasmonresonance using a SensiQ Pioneer (ICx Nomadics, Stillwater, Okla.) and acarboxylated COOH1 sensor (Ibid) amenable for amine coupling. Protein G(6510-10, Biovision, Mountain View, Calif.) was coupled to the COOH1sensor using amine coupling reagents (Sigma Aldrich(N-Hydroxysuccinimide (NHS, 56480),N-(3-Dimethylaminopropyl)-B′-ethylcarbodiimide hydrochloride (EDC,E7750), Ethanolamine (398136), St. Louis, Mo.) or with the Biacore AmineCoupling Kit (BR-1000-50, GE Healthcare, Waukesha, Wis.).

Briefly, the carboxylated surface is activated with 2 mM EDC and 0.5 mMNHS for a contact time ranging between 2-10 minutes. Protein G, invariable concentrations ranging between 20-400 ug/mL, was diluted into10 mM acetate buffer, pH 4.3 (sodium acetate, BP334-1; glacial aceticacid, A490-212; Thermo Fisher Scientific, Waltham, Mass.), and injectedover the activated sensor for variable contact times ranging between 5and 10 minutes at a rate ranging from 5-10 μL/min. Quantities of ProteinG immobilized to the COOH1 sensor chip range from 400-2000 responseunits (RU). Remaining activated sites were capped with 100 μL ofethanolamine at a flow rate of 25 μL/min.

Equilibrium constants for rabbit human chimeric mAbs were determined bybinding the mAb to the protein G coated chip followed by binding of eachanalyte (IL-6 or IL-23) to its respective mAb. In order to minimize masstransfer effects, the surface densities of the mAbs for each analytewere adjusted so that as analyte binding approached saturation itscorresponding RU fell between 200 and 300. Dilutions of 3×-FLAG-IL-6(see example 1), IL-6 (CYT-213, Prospec-Tany Technogene, Rehovot,Israel) or human dimeric IL-23 (34-8239, eBiosciences, San Diego,Calif.) ranging from 1 to 100 nM were injected over the chip surface andassociation (K_(a)) and dissociation (K_(d)) rate constants weremeasured. For each binding and dissociation cycle the chip surface wasregenerated with 15 uL of 20 mM NaOH (5671-02, Mallinckrodt Baker,Philliphsburg, N.J.). The assay temperature was maintained at 25° C.with an analyte flow rate of 50 μL/min and included a 2 minuteassociation phase and 10-30 minute dissociation phase. The on/off rates(k_(a)/k_(d)) and dissociation constants (K_(D)) were determined usingthe format described above along with pseudo-first-order 1:1 interactionmodel software (Qdat, ICx Nomadics, Stillwater, Okla.).

Equilibrium constants for scFvs were determined as previously described;however, the protocol was modified so that an epitope tagged IL-6 wascaptured on the chip surface and the dissociation of anti-IL-6 scFvsfrom IL-6 was monitored. Briefly, anti-FLAG® M2 antibody (200472,Agilent Technologies, Santa Clara, Calif.) was bound to Protein G, andthen 3×FLAG-IL-6 was captured by the anti-FLAG antibody. The anti-IL-6scFvs were assayed over a range of concentrations between 1 and 100 nM.

SPR of bispecific scFvs were carried out as previously described formeasuring the anti-IL-6 moiety; however, protocol modifications wererequired in order to determine the binding kinetics of the anti-IL-23scFv, 31A12. Briefly, IL-23 binding by the bispecific was performed byfirst immobilizing the bispecific with IL-6 as described, but at aconstant density (˜240 RU). Binding and dissociation of a recombinanthuman dimeric IL-23 (34-8239, eBiosciences, San Diego, Calif.) wasassayed in concentrations ranging from 3 to 25 nM using the sameparameters detailed above.

Example 3 Generation of Rabbit Anti Human IL-6 Monoclonal Antibodies 3.1Rabbit Immunization:

One New Zealand White rabbit was immunized with 100 μg of IL-6 protein(Recombinant E. Coli-derived human IL-6, Ref. Seq. accession NP000591.1,obtained from ProSpec-Tany TechnoGene Ltd., Rehovot, Israel (Cat. #CYT-213i)) in Sigma Adjuvant System (Sigma S6322), at days 0, 21, and42. The rabbit was boosted not less than 10 days prior to bleeding.Rabbits were maintained at R & R Research Laboratories (Stanwood, Wash.,USA) in accordance with NIH, USDA and IACUC guidelines.

3.2 B Cell Cloning:

30 ml of blood were harvested from each rabbit by venipuncture.Peripheral Blood Mononuclear cells (PBMC) were prepared by densitycentrifugation (Lympholyte-rabbit, Cat. # CL5050, Cedarlane LaboratoriesLtd., Ontario, Canada).

The neutralizing activity of immune rabbit serum against human IL-6 wasassayed after 3 immunizations.

Human IL-6 is titered from 1 ng/ml with and without a 1:3200 dilution ofimmune rabbit serum. To select B-cells specific for IL-6, 6 cm tissueculture petri dishes were coated with IL-6 as follows: His-tagged humanIL-6 (Cat. # CYT-484, Prospec-Tany Technogene, Rehovot, Israel) wascaptured on an anti-His antibody coated plate. Anti-His antibody (Cat #A00613, GeneScript Corp., New Jersey, USA) at 2 μg/ml in PBS, wasincubated overnight at 4 C or 1 hour at 37 C in a 6 cm plastic petridish. The antibody solution was then removed and 4 ml of PBS+5% BSA wasadded for 1 hour. IL-6 was captured by incubating 3 ml of His-IL-6 at 2μg/ml in PBS for 1 hour, followed by 4 washes with PBS. PBMC weresuspended in 2 ml of PBS containing 5% BSA, and plated on theantigen-coated dishes for 40 minutes at 4 C. The plates weresubsequently washed 4 to 8 times with PBS, and the adherent cells wereremoved by gentle scraping. These cells were resuspended in completemedium (RPMI 1640, 10% FBS, Non-Essential Amino Acids, Pyruvate, 50 uMbeta-mercaptoethanol) at 100 to 500×10³ cells/ml. 100 μl of cellsuspension was added to each well of a 96-well plate, in addition to 100μl complete medium containing Mitomycin-c treated EL4-B5 cells (Zubleret al 1985) at 5×10⁵ cells/well, recombinant human IL-2 (GenScript Corp,Piscataway, N.J., USA) at 20 ng/ml, and 5% conditioned media from rabbitspleen cells.

Briefly, EL4-B5 cells were suspended at a density of 1×107 cells/ml inRPMI containing 50 μg/ml mitomycin-c (Cat # M0503, Sigma-Aldrich) for 40minutes and washed 6 times in complete medium.

The rabbit conditioned media was prepared as follows: Rabbit spleencells were mechanically dissociated, filtered through a 70 μm mesh,resuspended at 1×10⁶ cells/ml, in complete medium (RPMI 1640, 10% FBS,Non-Essential Amino Acids, Pyruvate, 50 uM beta-mercaptoethanol). Thecells were stimulated with 500 ng/ml of lonomycin and eitherConcanavalin A (Cat # C 5275, Sigma-Aldrich) at 5 μg/ml or PMA at 40ng/ml for 48 h in a CO2 (5%) incubator at 37° C. The conditioned mediumwas sterile filtered and stored at −20 C for subsequent experiments. Forsome experiments mitomycin-c treated (as per EL4-B5) normal rabbitspleen cells were added to the cloning plates at 1−2×10⁵ cells/well.

The plates containing antigen-selected cells were incubated for 7 to 10days at 37° C., 5% CO2. The culture supernatants were then harvested tobe assayed for IL-6 binding (ELISA) as well as inhibition of IL-6activity.

An ELISA was used to evaluate IL-6 binding. Recombinant IL-6 (SeeExample 1) was added to an ELISA plate in 100 μl PBS at 0.25 μg/ml.Plates were incubated 1 hour at 37 C, or overnight at 4 C. To block 100μl/well PBS containing 10% goat serum (Cat #16210-072, Invitrogen, USA)was added to each well. Plates were incubated 1 hour at roomtemperature. Plates were rinsed 5 times with de-ionized water. To eachwell was added 50 μl PBS/10% goat serum. Test samples were then added at50 μl/well. Plates were incubated 1 hour at room temperature. Plateswere rinsed 5 times with de-ionized water. To each well was added 100 μlperoxidase-conjugated goat anti-rabbit IgG (Cat. #111-035-008, JacksonImmuno Research) diluted 1:5000 in PBS/10% goat serum. Plates wereincubated 1 hour at room temperature, then washed 5 times withde-ionized water. TMB substrate (Thermo Scientific, Rockford, Ill., USA)was added at 100 μl/well. Reaction was stopped with 100 μl 1N H₂SO₄ (JTBaker, Phillipsburg, N.J., USA). Absorbance was measured at 450 nm usinga Molecular Devices M2 plate reader.

A bioassay using an IL-6 dependant murine B-cell hybridoma cell line(B9cell line; Aarden et al., 1987) was used to evaluate IL-6 inhibition,by means of measuring B9 cell proliferation in response to E. Coli orCHO cell derived human IL-6 (Prospec-Tany Technogene, Rehovot, Israel)Samples to be tested for neutralizing activity were diluted in 100 μlassay medium (RPMI 1640 w/L-glutamine, 10% FBS, Non-Essential AminoAcids, Sodium Pyruvate, 50 μM 2-mercaptoethanol) in a 96-well tissueculture plate. This was followed by the addition of 50 μl of IL-6 (Cat.# CYT-274 Prospec-Tany Technogene) containing assay medium, and 30minutes of incubation at room temperature. B9 cells were recovered fromflasks and centrifuged for 7 min at 180×g, and the pellet wasresuspended in IL-6-free culture medium (RPMI 1640 w/L-glutamine, 10%FBS, Non-Essential Amino Acids, Sodium Pyruvate, 50 μM2-mercaptoethanol). Cells were centrifuged and resuspended three timesto remove IL-6. Following viability determination by trypan blueexclusion, cells were adjusted to 1×10⁵ cells/ml. A volume of 50 μl ofB9 cells, corresponding to 5×10³ cells, was added to each well alongwith appropriate control wells containing IL-6-free medium.

The plates were incubated for 48 h at 37° C. 5% CO2. Subsequently, 20 μlof Alamar Blue (Cat # DAL1100, Invitrogen, USA) was added to each well,and the plates were incubated for an additional 18 h. The plates wereread on a Molecular Devices (Sunnyvale, Calif., USA) M2 plate reader at570 and 600 nm.

FIG. 1 illustrates an exemplary experiment as carried out to selectcells producing antibodies suitable for further characterization: eachsupernatant was tested for both IL-6 binding (lower panel) and IL-6neutralization (upper panel). Supernatants suitable for furthercharacterization were positive in both assays (arrow and star).

3.3 V-Region Rescue from Rabbit B-Cells

The V-region rescue process is summarized in FIG. 2.

Briefly, IgG variable heavy and light chains from the supernatantspositive for both IL-6 neutralization and IL-6 binding tests werecaptured by amplification using reverse transcriptase coupled polymerasechain reaction (RT-PCR). The VH and VL cDNAs thus obtained, were clonedand ligated onto human constant region constructs, such that the finalcDNA construct encoded a chimeric rabbit human IgG as shown in FIG. 3.

Primers for rescuing immunoglobulin V-regions from activated rabbitB-cells were designed for both cDNA synthesis from mRNA captured aftercell lysis as well as the subsequent PCR amplification steps in whichthe final PCR adds restriction enzyme sites for cloning into anexpression vector. Since one rabbit would have a b9 allotype background(Rader et al, 2000), it was necessary to design various cDNA primers andnested J-region primers as J-kappa and Ckappa region usage differbetween rabbit allotypes (Sehgal et al., 1990). A list of selected DNAoligonucleotide primers used in both the RT and PCR steps is shown inTable 1, with their heavy/light chain specificity designated in theright hand column.

Selected positive B-cells were lysed and mRNA prepared using the mRNADIRECT Micro Kit, from Dynabeads (Cat. #610.21) according to themanufacturer's instructions. To recover the v-regions, mRNA generatedfrom a single antigen positive well is used in a one-step RT/PCR (QiagenOne Step RT-PCR Kit, cat. N. 210212) reaction for both the heavy andlight chains. For the reactions, gene specific primers located in theconstant regions of the heavy and light chains of the rabbit IgGmolecule are used to generate a single strand cDNA, followed by nestedJ-region primers together with Leader peptide-specific primers for firstround PCR generation

TABLE 1 Primer sets for V-region rescue of the rabbit variable-regions.Primer Specificity SEQ Primer (Heavy Chain/Light ID name Sequence 5′-3′Chain) RT/1st Rnd 66 rCH1R1 GCCAGTGGGAAGACTGACGGAG H 67 rVHL-FATGGAGACTGGGCTGCGCTGG H 68 rJH-R1 GGAGACGGTGACCAGGGTGCCTGGG H 69 rJH6RTGAAGAGACGGTGACGAGGGTC H 70 rCk1R1 GCAGCTGGTGGGAAGATGAGGAC L 71 rVK5UTRGCCAGGCAGGACCCAGCATGGAC L 72 rJK2-R ACCACCACCTYGGTCCCTCCGCC L 73 rJK1-RGATTTCYACCTTGGTGCCAGCTCC L 74 rJK24R GTTTGATCTCCACCTTGGTCCCCGCACCG L 75rJK2b9R ACTTACATAGGATCTCCAGCTC L 76 rJK5R GTTTGATCTCCAGCTTGGTTCC L2nd Rnd 77 rVH1a-H3F GCGATAAGCTTCACCATGGAGACTGGGCTGCGCTGG H 78rJH-XhoR ii GCGATCTCGAGACGGTGACCAGGGTGCCTGGG H 79 rJH6XholRGCATAGCTCGAGGAGACGGTGACGAGGGTCCCTG H 80 rVKF-NcoGCGATACCATGGACACGAGGGCCCCCACTCAGCTG L 81 rJK2-BsiR2GCGAACGTACGGACSACCACCACCTYGGTCCCTCCGCC L 82 rJK1-BsiR2GCGAACGTACGTTTGATTTCYACCTTGGTGCCAGCTCC L 83 rJK24BsiRGCATACGTACGTTTGATCTCCACCTTGGTCCCCGCACCG L 84 rJK2b9BsiRGCATACGTACGTAGGATCTCCAGCTCGGTCCC L 85 rJK5BsiRGCATACGTACGTTTGATCTCCAGCTTGGTTCC L

A second round of PCR is performed to add restriction sites to therescued V-regions for subcloning into vectors containing the constantregions of either the heavy or light chain of human IgG1. Separate PCRsare performed for heavy and light chains. Restrictions sites added tothe V-regions are HindIII/XhoI and NcoI/BsiW1 for heavy and light chainsrespectively. Vectors containing constant regions were obtained fromInvivoGen (pFUSE-CHIg-hG1 #08E07-SV and pFUSE2-CLIg-hk #08F19-SV). Bothvectors were modified in-house for the sub cloning strategy. Afteraddition of the restriction sites, the PCR products were subjected tothe relevant Restriction enzymes digestion, gel purified and ligatedinto the appropriate vector.

Following sub cloning, the ligated DNA was transformed into DH5α E.coli. (Invitrogen). The entire transformation mixture was cultured overnight in medium containing the appropriate antibiotic resistance. Thecultured bacteria were harvested and plasmid DNA was isolated andpurified (Qiagen kit) for use in transient HEK293 expression of chimericantibodies. At this time the isolated DNA may or may not be homogenousfor one specific V-region, as selected wells may contain one or moredifferent B-cell clones. To generate the chimeric antibodies, HEK293cells were co-transfected with the DNA of both heavy and light chainfrom a selected well. Supernatant was harvested after three to five daysof cell culture and assayed for IgG and antigen binding by ELISA., aswell as IL-6 neutralization (see above for methods). To detect thepresence of IgG in the transfection supernatant, an ELISA immunoassay isdone which utilizes an anti-human IgG Fc capture antibody coated to anELISA plate, followed by the supernatants and human IgG standard.Detection of Fc-captured antibody is obtained using an anti-human IgG(H&L)-HRP reagent and TMB substrate.

DNA sequencing was used to screen each ELISA-positive well to determinehow many unique heavy and light chain combinations were rescued underthe assumption that there would likely be more than one unique clonepresent per well. For DNA sequencing, the DNA isolated previously fortransfection is retransformed into DH5α E. coli and plated on agarplates containing the appropriate antibiotic. Multiple colonies fromeach transformation are picked and processed for DNA production using arolling circle DNA amplification kit (Templiphy, GE Healthcare)following manufacturer's instructions. The DNA generated from theTempliphy reactions is sequenced and subsequently analyzed to determinethe complexity of V-regions for each well. In addition to making DNA,bacteria used for the Templiphy reactions are saved for future DNAisolation since each DNA now represents a unique clone.

Following sequence analysis, and using the bacteria saved from theTempliphy reactions, DNA was obtained for each unique V-region for boththe heavy and light chains from each rescued well. Heavy and lightchains were matched and transiently transfected into HEK293. When morethan one possible heavy and light chain combination was present (wellsnot clonal), every possible combination of unique heavy and light chainpairs were transfected. Supernatants were harvested after three to fivedays , assayed for IgG and antigen binding by ELISA, as well as IL-6neutralization (see above for methods). After this deconvolution step,heavy and light chain combinations which retained the desired activitywere selected for humanization.

The following antibody clones met the criteria for antigen binding andantigen neutralization and were selected for further development:

13A8:

Variable region Heavy Chain (Vh) identified as SEQ ID 6, aminoacidsequence ; SEQ ID 7, nucleotide sequence;Variable region Light chain (VI) identified as SEQ ID 8, aminoacidsequence; SEQ ID 9, nucleotide sequence.

The 13A8 clone demonstrated high potency, with an EC50 (calculated asconcentration necessary to inhibit bioactivity of 50 pg/ml of IL-6) of34 pg/ml (FIGS. 3A and B), and high affinity antigen binding propertiesdetermined by SPR analysis: K_(d) 1.38×10⁻⁴ (s⁻¹); K_(a) 6.33×10⁵(M⁻¹s⁻¹), and K_(D) 218 pM

TABLE 2 ID SEQ ID Sequence 13A8 VH- CDR1 10 CDR1 VH SYDMS 13A8 VH- CDR211 CDR2 VH YIYTDSSTWYANWAKG 13A8 VH- CDR3 12 CDR3 VH GSTDYAFDTRLDL13A8 VK- CDR1 13 CDR1 VL QASQSISNELS 13A8 VK- CDR2 14 CDR2 VL RASTLTS13A8 VK- CDR3 15 CDR3 VL QQGYNSNDVDNV

28D2

Variable region light chain (Vh) identified as SEQ ID 16, aminoacidsequence and SEQ ID 17, nucleotide sequence.

Variable region Light chain (VI) identified as SEQ ID 18, aminoacidsequence and SEQ ID 19, nucleotide sequence.

The 28D2 clone demonstrated high potency, with an EC50 (calculated asconcentration necessary to inhibit bioactivity of 50 pg/ml of IL-6) of65 pg/ml (FIGS. 3A and B).

TABLE 3 ID SEQ ID Sequence 28D2 VH- CDR1 20 CDR1 VH KNAIA 28D2 VH- CDR221 CDR2 VH IIYAGGATTYASWAKG 28D2 VH- CDR3 22 CDR3 VH EYAGDSYYTGYTQLD28D2 VK- CDR1 23 CDR1 VL QASEDLFSSLA 28D2 VK- CDR2 24 CDR2 VL SASTLAS28D2 VK- CDR3 25 CDR3 VL LGLYYYLTPDPIYG

18D4

Variable region light chain (Vh) identified as SEQ ID 26, aminoacidsequence and SEQ ID 27, nucleotide sequence.

Variable region Light chain (VI) identified as SEQ ID 28, aminoacidsequence and SEQ ID 29, nucleotide sequence

The 18D4 clone demonstrated high potency, with an EC50 (calculated asconcentration necessary to inhibit bioactivity of 50 pg/ml of IL-6) of54 pg/ml, and high affinity antigen binding properties determined by SPRanalysis: K_(d) 8.49×10⁻⁵ (s⁻¹); K_(a) 6.66×10⁵ (M⁻¹s⁻¹), and K_(D) 128pM

TABLE 4 ID SEQ ID Sequence 18D4 VH- CDR1 30 CDR1 VH SYAMT 18D4 VH- CDR231 CDR2 VH TSYVYSGDTWYASWVKG 18D4 VH- CDR3 32 CDR3 VH VGDYDDYGAHDVFDS18D4 VK- CDR1 33 CDR1 VL QASESISSWLS 18D4 VK- CDR2 34 CDR2 VL RASTLAS18D4 VK- CDR3 35 CDR3 VL QQGYTGGNVDNA

8C8

Variable region light chain (Vh) identified as SEQ ID 36, aminoacidsequence and SEQ ID 37, nucleotide sequence.

Variable region Light chain (VI) identified as SEQ ID 38, aminoacidsequence and SEQ ID 39, nucleotide sequence

The initial transfection supernatants of the 8C8 clone demonstrated highpotency for inhibition of bioactivity. Subsequently, scFv were deriveddirectly from the rabbit IgG and these demonstrated high potency, withan EC50 (calculated as concentration necessary to inhibit bioactivity of200 pg/ml of IL-6) of 510 pg/ml (FIG. 12A).

TABLE 5 ID SEQ ID Sequence 8C8 VH- CDR1 40 CDR1 VH SSGVS 8C8 VH- CDR2 41CDR2 VH YVSIADTISYANWAKG 8C8 VH- CDR3 42 CDR3 VH GFITYSGVL 8C8 VK- CDR143 CDR1 VL QASQSISNELS 8C8 VK- CDR2 44 CDR2 VL RTSTLAS 8C8 VK- CDR3 45CDR3 VL QQGYNSNDVDNV

9H4

Variable region light chain (Vh) identified as SEQ ID 46, aminoacidsequence and SEQ ID 47, nucleotide sequence.

Variable region Light chain (VI) identified as SEQ ID 48, aminoacidsequence and SEQ ID 49, nucleotide sequence

The 9H4 clone demonstrated high potency, with an EC50 (calculated asconcentration necessary to inhibit bioactivity of 50 pg/ml of IL-6) of109 pg/ml (FIGS. 3C, D, E), and high affinity antigen binding propertiesdetermined by SPR analysis: K_(d) 4.75×10⁻⁵ (s⁻¹); K_(a) 8.16×10⁵(M⁻¹s⁻¹), and K_(D) 58 pM

TABLE 6 ID SEQ ID Sequence 9H4 VH- CDR1 50 CDR1 VH SYDMS 9H4 VH- CDR2 51CDR2 VH YIYTDTSTYYANWAKG 9H4 VH- CDR3 52 CDR3 VH GSTDYAFDTRLDL9H4 VK- CDR1 53 CDR1 VL QASQSISNELS 9H4 VK- CDR2 54 CDR2 VL RTSTLAS9H4 VK- CDR3 55 CDR3 VL QQGYNSNDVDNV

9C8

Variable region light chain (Vh) identified as SEQ ID 56, aminoacidsequence and SEQ ID 57, nucleotide sequence.

Variable region Light chain (VI) identified as SEQ ID 58, aminoacidsequence and SEQ ID 59, nucleotide sequence

The 9C8 clone demonstrated high activity and antigen binding properties,with an EC50 (calculated as concentration necessary to inhibitbioactivity of 50 pg/ml of IL-6) of 400 pg/ml (FIG. 3E), and K_(d)3.17×10⁻⁵ (s⁻¹); K_(a) 7.65×10⁵ (M⁻¹s⁻¹), and K_(D) 42 pM

TABLE 7 ID SEQ ID Sequence 9C8 VH- CDR1 60 CDR1 VH SYDMS 9C8 VH- CDR2 61CDR2 VH YIYTDSSTYYANWAKG 9C8 VH- CDR3 62 CDR3 VH GSTDYAFDTRLDL9C8 VK- CDR1 63 CDR1 VL QASQSISNELS 9C8 VK- CDR2 64 CDR2 VL RTSTLAS9C8 VK- CDR3 65 CDR3 VL QQGYNSNDVDNV

3.4 Reactivity Against Primate IL-6

The successful development of any cytokine antagonist for human therapywill require initial toxicology testing. Toxicology is most efficientlydemonstrated in non human species. In order to conduct toxicologystudies it is first necessary to demonstrate the capacity of theantibodies to neutralize IL-6 from the species being considered for thestudies.

Several of the anti-IL6 chimeric antibodies that have been tested forneutralization of non-human primate IL-6 activity are shown in FIG. 3.

Example 4 Generation of Rabbit Anti Human IL-23 Monoclonal Antibodies4.1 Rabbit Immunization

One New Zealand White (NZW) and one B9 rabbit were immunized with 100 μgof IL-23 protein (baculovirus-derived recombinant human IL-23 composedof the p40 chain, accession NM_(—)002187 and the p19 chain, accessionNM_(—)016584, from eBiosciences, San Diego Calif., USA (Cat. #34-8239))in Sigma Adjuvant System (Sigma S6322), at days 0, 21, and 42. Theanimals were bled at least 10 days after immunization. Rabbits weremaintained at R & R Research Laboratories (Stanwood, Wash., USA) andSpring Valley Laboratories (Woodbine, Md., USA) in accordance with NIH,USDA and IACUC guidelines.

The NZW and B9 rabbits express different immunoglobulin gene allotypeswhich correspond to differences in the framework and CDR regions andcorresponding differences in the structures of the mAbs isolated. The b9allotype rabbits are reported to be better for use in phage displaycloning of mAbs, due to the absence of Cys residues in the V region. Allthe anti IL-6 mAbs were cloned from NZW rabbits, and none presented Cysresidues in the V region.

The IL-23 neutralization activity of high titer sera from B9 and NZWrabbits immunized with human IL-23 was measured. Serum from immunerabbits is able to fully neutralize the IL-17A secretion induced by 600pg/ml of human IL-23 from mouse splenocytes at dilutions approaching1:10,000.

4.2 B Cell Cloning

B-Cells specific for IL-23 were selected as in Example 3.

Peripheral Blood Mononuclear cells (PBMC) were prepared by densitycentrifugation (Lympholyte-rabbit, Cat. # CL5050, Cedarlane LaboratoriesLtd., Ontario, Canada) from each rabbit. IL-23 coated plates wereproduced by incubating IL23 (eBioscience) at 2 μg/ml in PBS overnight at4 C, or 1 hour at 37 C, washed 4 times with PBS and used for capturingB-cells. PBMC were suspended in 2 ml of PBS containing 5% BSA, andplated on the antigen-coated dishes for 40 minutes at 4 C. The plateswere subsequently washed 4 to 8 times with PBS, and the adherent cellswere removed by gentle scrapping. Cells were plated in rabbit spleencells conditioned medium and EL4-B5 cells (see example 1) and incubatedat 37° C., 5% CO2 for seven to ten days. The culture supernatants werethen harvested and tested for IL-23 binding (ELISA) and inhibition ofIL-23 activity

An ELISA assay was used to evaluate IL-23 binding (Aggarwal et al.,2003). ELISA plates were coated using either a direct or indirect methodof binding IL-23.

For the indirect binding method anti-His antibody (Cat # A00613,GenScript Corp., New Jersey, USA) was added to the plates in 100 μl/wellof PBS at 0.01-0.02 ug/ml. Plates were incubated 1 hour at 37 C, orovernight at 4 C. To block non-specific binding 100 μl/well PBScontaining 10% goat serum (Cat #16210-072, Invitrogen, USA) was added toeach well, after which plates were rinsed 5 times with de-ionized water.IL-23 p40-p19-His (SEQ ID 4) in 100 μl/well PBS/10% goat serum at 0.5μg/ml was added and incubated for 1 hour at room temperature.

For the direct binding method IL-23 p40-p19-His (SEQ ID 4) was added toan ELISA plate in 100 μl PBS at 0.5 μg/ml. Plates were incubated 1 hourat 37 C, or overnight at 4 C. To block non-specific binding 100 μl/wellPBS containing 10% goat serum (Cat #16210-072, Invitrogen, USA) wasadded to each well. Plates were incubated 1 hour at room temperature.

After IL-23 binding, plates were rinsed 5 times with de-ionized water.To each well was added 50 μl PBS/10% goat serum. Test samples were thenadded at 50 μl/well. Plates were incubated 1 hour at room temperatureand were rinsed 5 times with de-ionized water. To each well was added100 μl peroxidase-conjugated goat anti-rabbit IgG (Cat. #111-035-008,Jackson Immuno Research) diluted 1:5000 in PBS/10% goat serum. Plateswere incubated 1 hour at room temperature, then washed 5 times withde-ionized water. TMB substrate (Thermo Scientific, Rockford, Ill., USA)was added at 100 μl/well. The reaction was stopped with 100 μl 1N H₂SO₄(JT Baker, Phillipsburg, N.J., USA). Absorbance was measured at 450 nmusing a Molecular Devices M2 plate reader.

A bioassay, based on the detection of IL-23-induced IL-17 expression bymouse spleen cells, was used to detect antibody mediated inhibition ofIL-23 binding to the IL-23 receptor and resulting bioactivity.

5×10⁵ C57Bl/6 spleen cells were cultured in the wells of a 96-well platein 200 μl containing a dilution of the heterodimeric IL-23 (eBiosciencecat. #14-8239 or Humanzyme, Chicago, USA cat. #HZ-1049) and the platesincubated for 2-3 days at 37 C. Culture medium is RPMI 1640, 10% FBS, 50uM 2-mercaptoethanol, non-Essential Amino Acids, pyruvate, gentamicinand 10 ng/ml human IL-2 (Cat # CYT-209, Prospec-Tany Technogene). After3 days, the culture supernatants were assayed by ELISA for IL-17A, asdescribed below.

To assay for IL-23 inhibition, test mAb samples were added at variousdilutions to the cultures of mouse spleen cells containing 150-1200pg/ml IL-23 and the secretion of IL-17A was compared to cultures nottreated with mAb.

An ELISA assay was used to detect mouse IL-17. Plates were coated withanti-mlL-17A (eBioscience #14-7178) 1 μg/ml in 100 μl PBS, overnight at4° C. or 1 hr at 37° C. Plates were washed in deionized water andblocked for 1 h with 100 μl of PBS, 10% goat serum. After washing theplates, 50 μl of PBS/10% goat serum and 50 μl of culture supernatantwere added to the plates, and incubated for 1 hr. The plates were washedand 100 μl/well of anti-mIL-17A-Biotin (eBioscience #13-7179) at 0.5μg/ml in PBS/10% goat serum was added and the plates were incubated for1 h at RT, washed, and reacted with 100 μl/well Streptavidin-HRP(Jackson Labs) at 1:1000 in PBS/10% goat serum. Plates were washedagain, and the signal was detected by adding Add 100 μl/well TMBsubstrate (Thermo Scientific, IL, USA). After stopping the reaction with100 μl/well 1N H₂SO₄, the optical density was read at 450 nM. Data wereplotted and analyzed with Graphpad (Prism, Mountainview, Calif.)software.

B cells were cloned from the IL-23 immunized rabbits and the B cellclone supernatants were tested for IL-23 neutralization and IL-23binding.

FIG. 7 illustrates an example 96 well plate from an experiment whereeach supernatant was tested for both IL-23 binding (lower panel) andIL-23 neutralization (upper panel). Supernatants suitable for furthercharacterization were positive in both tests.

4.3 V-Region Rescue from Activated B-Cells:

The IgG variable heavy and light chains from the B cells positive forboth IL-23 neutralization and IL-23 binding assays were captured byRT-PCR essentially as in Example 3.

FIGS. 5A-I show examples of human IL-23 neutralization activity byseveral anti IL-23 neutralizing mAbs obtained.

Several antibodies have been further characterized in their binding toprimate IL-23, as depicted in FIG. 6. Monoclonal antibodies neutralizingIL-23 were further tested for neutralization of human IL-12, as shown inFIG. 8A. mAb 31A12 neutralizes specifically IL-23 while 45G5 and 22H8neutralize both IL-23 and IL-12.

Mapping of epitopes recognized by antibodies of the present inventionmay be achieved through several experimental methods, such as crosscompetition binding assays, or binding to linear peptides.

Detailed epitope mapping can be obtained through cocrystallization ofthe monoclonal antibody or antibody fragment thereof and antigencomplex. An alternative method uses Liquid Chromatography MassSpectroscopy (LCMS) analysis of antigen peptides after labeling withdeuterium in the presence of the mAb. Non deuterated residues representthose protected by the mAb.

The following monoclonal antibodies having met the criteria for antigenbinding, antigen neutralization and selective binding of IL-23, wereselected for further development:

31A12:

Variable region Heavy Chain (Vh) identified as SEQ ID 86, aminoacidsequence ; SEQ ID 87, nucleotide sequence;

Variable region Light chain (VI) identified as SEQ ID 88, aminoacidsequence; SEQ ID 89, nucleotide sequence.

The 31A12 mAb demonstrated high potency and antigen binding properties,with an EC50 (calculated as concentration necessary to inhibitbioactivity of 600 pg/ml of IL-23) of 3286 pg/ml, and high affinityantigen binding properties determined by SPR analysis: K_(d) 2.02×10⁻⁴(s⁻¹); K_(a) 4.79×10⁵ (M⁻¹s⁻¹), and K_(D) 422 pM.

TABLE 8 ID SEQ ID Sequence 31A12 VH CDR1 90 CDR1 VH SYWMT 31A12 VH CDR291 CDR2 VH TIATSSTYYASWAKG 31A12 VH CDR3 92 CDR3 VH GLTTDYDLDL31A12 VK CDR1 93 CDR1 VL QASEDIESYLA 31A12 VK CDR2 94 CDR2 VL SASTLTS31A12 VK CDR3 95 CDR3 VL LGADDTTTV

49B7

Variable region light chain (Vh) identified as SEQ ID 96, aminoacidsequence and SEQ ID 97, nucleotide sequence.

Variable region Light chain (VI) identified as SEQ ID 98, aminoacidsequence and SEQ ID 99, nucleotide sequence.

The 49B7 mAb demonstrated high potency, with an EC50 (calculated asconcentration necessary to inhibit bioactivity of 600 pg/ml of IL-23) of988 pg/ml.

TABLE 9 ID SEQ ID Sequence 49B7 VH CDR1 100 CDR1 VH DYDMS 49B7 VH CDR2101 CDR2 VH IVYDIGTIYYAPWAEG 49B7 VH CDR3 102 CDR3 VH EAPGYSDGDI49B7 VK CDR1 103 CDR1 VL QASETVDNNKRLS 49B7 VK CDR2 104 CDR2 VL GAATLAS49B7 VK CDR3 105 CDR3 VL GGYKDSTDVG

16C6

Variable region light chain (Vh) identified as SEQ ID 106, aminoacidsequence and SEQ ID 107 nucleotide sequence.

Variable region Light chain (VI) identified as SEQ ID 108, aminoacidsequence and SEQ ID 109, nucleotide sequence

The 16C6 mAb demonstrated high potency, with an EC50 (calculated asconcentration necessary to inhibit bioactivity of 150 pg/ml of IL-23) of219 pg/ml.

TABLE 10 ID SEQ ID Sequence 16C6 VH CDR1 110 CDR1 VH TVSGFSLNSYSMS16C6 VH CDR2 111 CDR2 VH VIGLGGSAYYASWAK 16C6 VH CDR3 112 CDR3 VHATYSDDNI 16C6 VK CDR1 113 CDR1 VL QASQSISSWLS 16C6 VK CDR2 114 CDR2 VLRASTLAS 16C6 VK CDR3 115 CDR3 VL LGGDGNVSN

34E11

Variable region light chain (Vh) identified as SEQ ID 116, aminoacidsequence and SEQ ID 117, nucleotide sequence.

Variable region Light chain (VI) identified as SEQ ID 118, aminoacidsequence and SEQ ID 119, nucleotide sequence

The 34E11 clone demonstrated high potency, with an EC50 (calculated asconcentration necessary to inhibit bioactivity of 150 pg/ml of IL-23) of50 pg/ml.

TABLE 11 ID SEQ ID Sequence 34E11 VH CDR1 120 CDR1 VH TYDIN34E11 VH CDR2 121 CDR2 VH YIYRGSPYYADWAKG 34E11 VH CDR3 122 CDR3 VHNLYSVNVL 34E11 VK CDR1 123 CDR1 VL QASQSISSRLA 34E11 VK CDR2 124 CDR2 VLSASTLAS 34E11 VK CDR3 125 CDR3 VL LGSYSNTIRT

35H4

Variable region light chain (Vh) identified as SEQ ID 126, aminoacidsequence and SEQ ID 127, nucleotide sequence.

Variable region Light chain (VI) identified as SEQ ID 128, aminoacidsequence and SEQ ID 129, nucleotide sequence.

The 35H4 clone demonstrated high potency in the transfectionsupernatants of the cloned mAb relative to other mAbs isolated in thesame B cell cloning experiment (FIG. 5I).

TABLE 12 ID SEQ ID Sequence 35H4 VH CDR1 130 CDR1 VH DYDMS 35H4 VH CDR2131 CDR2 VH IVYDIGTIYYAPWAEG 35H4 VH CDR3 132 CDR3 VH EAPGYSDGDI35H4 VK CDR1 133 CDR1 VL QASETVGNNNRLS 35H4 VK CDR2 134 CDR2 VL GAATLAS35H4 VK CDR3 135 CDR3 VL GGYKDSTDVG

Example 5 Generation of Rabbit Anti Human IL-23/IL-12 MonoclonalAntibodies

As shown in FIG. 8A, it is possible to subdivide antibodies thatneutralize IL-23 into those that are IL-23 specific and those thatneutralize both IL-23 and IL-12. IL-12 and IL-23 share a common p40polypeptide and differ in the second chain, covalently linked to p40(FIG. 7A). The p19 chain of IL-23 and the p35 chain of IL-12 are bothfour helix bundle, cytokine like polypeptides. The p19 and p40 subunitsare linked to the common p40 subunit via a disulfide bond. Antibodiesneutralizing both IL-12 and IL-23 occur due to the sharing of the p40chain between the two molecules.

IL-12 and IL-23 receptors share a common chain (IL-12Rβ1) and inaddition, each have a unique receptor component (IL-23R and IL-12Rβ2)(FIG. 7B). These differences result in significant differences in thetarget cell and signaling pathways used by IL-12 and IL-23. Thesereceptors have transmembrane signaling domains that pair with JAK2 orTYK2 tyrosine kinases for STAT activation.

Antibodies binding both IL-23 and IL-12 were isolated from rabbitsimmunized with recombinant IL-23 (see Example 4). B cell clonesexhibiting binding and functional activity towards both IL-23 and IL-12were selected for further characterization.

Primary rescue transfections of chimeric IgG from B cells were testedfor neutralization of IL-23. Those that successfully neutralized IL-23were sequenced and subcloned and retransfected, and the mAbs quantitatedin the transfection supernatants. These mAbs were confirmed for antiIL-23 activity (FIG. 8B-E), and were then tested for neutralization ofIL-12 (FIG. 8F-G) and primate IL-23 (FIG. 8H, I).

The following monoclonal antibodies having met the criteria for antigenbinding, antigen neutralization and selective binding of IL-12 andIL-23, were selected for further development:

22H8:

Variable region Heavy Chain (Vh) identified as SEQ ID 136, aminoacidsequence; SEQ ID 137, nucleotide sequence;

Variable region Light chain (VI) identified as SEQ ID 138, aminoacidsequence; SEQ ID 139, nucleotide sequence.

mAb 22H8 demonstrated high potency and antigen binding properties, withan EC50 (calculated as concentration necessary to inhibit bioactivity of50 pg/ml of IL-6) of 603 pg/ml and high affinity antigen bindingproperties determined by SPR analysis: K_(d) 8.94×10⁻⁵ (s⁻¹); K_(a)4.03×10⁵ (M⁻¹s⁻¹), and K_(D) 221 pM.

TABLE 13 ID SEQ ID Sequence 22H8 VH CDR1 140 CDR1 VH TYTMN 22H8 VH CDR2141 CDR2 VH AISYDGGTAYANWAKG 22H8 VH CDR3 142 CDR3 VH GFYVYAYIGDAFDP22H8 VK CDR1 143 CDR1 VL QSSQTVYKNNLLS 22H8 VK CDR2 144 CDR2 VL LASTLAS22H8 VK CDR3 145 CDR3 VL LGGYDDDADTA

45G5:

Variable region Heavy Chain (Vh) identified as SEQ ID 146, aminoacidsequence ; SEQ ID 147, nucleotide sequence;

Variable region Light chain (VI) identified as SEQ ID 148, aminoacidsequence; SEQ ID 149 nucleotide sequence.

mAb 45G5 demonstrated high potency and antigen binding properties, withan EC50 (calculated as concentration necessary to inhibit bioactivity of50 pg/ml of IL-6) of 385 pg/ml.

TABLE 14 ID SEQ ID Sequence 45G5 VH CDR1 150 CDR1 VH VYPIN 45G5 VH CDR2151 CDR2 VH IINDVDDTAYSAWAKG 45G5 VH CDR3 152 CDR3 VH GYLSYAYAGDAFDP45G5 VK CDR1 153 CDR1 VL QSSQSIYNNNLLS 45G5 VK CDR2 154 CDR2 VL FASTLAS45G5 VK CDR3 155 CDR3 VL LGGYDDDADTA

1H1:

Variable region Heavy Chain (Vh) identified as SEQ ID 156, aminoacidsequence ; SEQ ID 157, nucleotide sequence;

Variable region Light chain (VI) identified as SEQ ID 158, aminoacidsequence; SEQ ID 159, nucleotide sequence.

mAb 1H1 demonstrated high potency and antigen binding properties, withan EC50 (calculated as concentration necessary to inhibit bioactivity of50 pg/ml of IL-6) of 603 pg/ml.

TABLE 15 ID SEQ ID Sequence 1H1 VH CDR1 160 CDR1 VH TASGLTLGSYSMT1H1 VH CDR2 161 CDR2 VH VIGVGGTLNYASWAQ 1H1 VH CDR3 162 CDR3 VH GTYSGDSI1H1 VK CDR1 163 CDR1 VL QASQSISSWLA 1H1 VK CDR2 164 CDR2 VL RASILTS1H1 VK CDR3 165 CDR3 VL LGGDGHVSN

4F3:

Variable region Heavy Chain (Vh) identified as SEQ ID 166 aminoacidsequence ; SEQ ID 167, nucleotide sequence;

Variable region Light chain (VI) identified as SEQ ID 168, aminoacidsequence; SEQ ID 169, nucleotide sequence.

mAb 4F3 demonstrated high potency and antigen binding properties, withan EC50 (calculated as concentration necessary to inhibit bioactivity of50 pg/ml of IL-6) of 2339 pg/ml.

TABLE 16 ID SEQ ID Sequence 4F3 VH CDR1 170 CDR1 VH GYTMI 4F3 VH CDR2171 CDR2 VH IISSSGNTYYASWVKG 4F3 VH CDR3 172 CDR3 VH GSGAYISDYFNV4F3 VK CDR1 173 CDR1 VL QASQSIDSWLS 4F3 VK CDR2 174 CDR2 VL SASKLAP4F3 VK CDR3 175 CDR3 VL QSYYDVNAGYG

5C5:

Variable region Heavy Chain (Vh) identified as SEQ ID 176, aminoacidsequence ; SEQ ID 177, nucleotide sequence;

Variable region Light chain (VI) identified as SEQ ID 178, aminoacidsequence; SEQ ID 179, nucleotide sequence.

mAb 5C5 demonstrated high potency and antigen binding properties, withan EC50 (calculated as concentration necessary to inhibit bioactivity of50 pg/ml of IL-6) of 1907 pg/ml.

TABLE 17 ID SEQ ID Sequence 5C5 VH CDR1 180 CDR1 VH SYTMI 5C5 VH CDR2181 CDR2 VH IISAGGSAYYASWVNG 5C5 VH CDR3 182 CDR3 VH GSGTYTSDYFNI5C5 VK CDR1 183 CDR1 VL QASQSIDSWLA 5C5 VK CDR2 184 CDR2 VL SASKLAT5C5 VK CDR3 185 CDR3 VL QSYYDANAGYG

14B5:

Variable region Heavy Chain (VH) identified as SEQ ID 186, amino acidsequence; SEQ ID 187, nucleotide sequence;

Variable region Light chain (VL) identified as SEQ ID 188, aminoacidsequence; SEQ ID 189, nucleotide sequence.

mAb 14B5 demonstrated high potency and antigen binding properties, withan EC50 (calculated as concentration necessary to inhibit bioactivity of50 pg/ml of IL-6) of 767 pg/ml.

TABLE 18 ID SEQ ID Sequence 14B5 VH CDR1 190 CDR1 VH DYTMI 14B5 VH CDR2191 CDR2 VH IISSSGNTYYATWVKG 14B5 VH CDR3 192 CDR3 VH GSGAYISDYFNV14B5 VK CDR1 193 CDR1 VL QASQSIDSWLS 14B5 VK CDR2 194 CDR2 VL AASKLAT14B5 VK CDR3 195 CDR3 VL QSYYDVNAGYG

5.1 IL-12 Bioassay

Antibodies were assayed for IL-12 neutralizing capacity using the IL-12responsive cell line NK-92 (CRL-2407, ATCC, Manassas, Va., USA). 50 μlof culture supernatant from the B cell cloning plates or 50 μl ofsupernatant from antibody transfection was transferred to a 96 welltissue culture plate. 50 μl of human IL-12 (Cat. # Cyt-362, Prospec-TanyTechnogene, Rehovot, Israel) was added to each well at 4 ng/ml. Plateswere incubated for 30-60 minutes at room temperature, after which 5×10⁴NK-92 cells were added to each well in 100 μl. Cultures were incubatedfor 3 days at 37 C, and supernatants assayed for humanInterferon-production. Assay medium is RPMI 1640, 10% FBS, NEAA,pyruvate, 50 μM 2-mercaptoethanol, gentamicin and 10 ng/ml human IL-2(Cat # Z00368, GeneScript Corporation, Piscataway, N.J., USA).

Interferon-γ ELISA

An ELISA assay was used to detect human Interferon-γ. Plates were coatedwith anti-human Interferon—(Cat. # Mab 1-D1K, Mabtech, Cincinnati, Ohio,USA) 1 μg/ml in 100μ PBS, overnight @4° C. or 1 hr @ 37° C. Plates werewashed in de-ionized water and blocked for 1 h with 100 μl of PBS, 10%goat serum. After washing the plates, 50 μl of PBS/10% goat serum and 50μl of culture supernatant were added to the plates, and incubated for 1hr. The plates were washed and 100 μl/well of anti-humanInterferon-γ-Biotin (Cat # Mab 7b6-1-biotin, Mabtech) at 0.5 μg/ml inPBS/10% goat serum was added and the plates were incubated for 1 h atRT, washed, and reacted with 100 μl/well Streptavidin-HRP (Jackson Labs)at 1:1000 in PBS/10% goat serum. Plates were washed again, and thesignal was detected by adding 00 μl/well TMB substrate (ThermoScientific, IL, USA). After stopping the reaction with 100 μl/well 1NH₂SO₄, the optical density was read at 450 nM.

Example 6 Engineering of Humanized scFvs Intro to Humanization

Rabbit immunoglobulin variable regions (V-regions) are captured frommRNA isolated from peripheral blood B-cells from immunized rabbits.These rabbit B-cells were plated at low density in 96-well plates andactivated as previously described. V-region cDNAs are amplified from themRNA of each well using reverse-transcriptase-PCR (RT-PCR) with agene-specific primer from the constant region for first strand synthesisand a nested J-region-specific primer at the 3′ end with a 5′ leaderprimer for the PCR step. V-regions are then cloned into either a humanIgG heavy chain, kappa or lambda light chain vector cassette ,transiently expressed in HEK 293 cells and 72-hour post-transfectionsupernatants tested for both total IgG expression and neutralization ofrespective targets. Potent neutralizers were then sequenced to determinethe level of complexity present in the well from which they weresubcloned. Once the number of unique light and heavy chains wasdetermined, all possible combinations present were again transientlyexpressed into HEK 293, assayed for neutralization and IgG content.Potent neutralizers were then further assayed for other desirableactivities. Anti-human IL-6 antibodies were tested for neutralization ofIL-6 from non-human primate. Anti-human IL-23 antibodies are assayed notonly for neutralization of non-human primate IL-23, but alsoneutralization of human IL-12 since both IL-12 and IL-23 dimers sharethe same p19 chain.

The inventors followed two strategies: they constructed rabbit scFvsdirectly from selected heavy chain (VH) and light chain (VK or VL) byPCR genetically fusing the heavy and light chain V-regions in either theVLVH or VHVL orientation by introducing a 20 amino acid linker composedof four tandem repeats of the sequence gly-gly-gly-gly-ser (G4S) betweenthe two domains. Rabbit scFvs are helpful in assessing whether or notthe conversion from a chimeric antibody to an scFv format has had anadverse effect on the functional or biophysical properties of theV-region pair. ScFvs were then transiently expressed in HEK 293 andassayed for function as illustrated in FIG. 12A. Potent neutralizerswere selected for humanization (see section 6.1)

Alternatively, Rabbit V-regions were humanized directly in an scFvformat (see below, 6.2)

Immunoglobulin V-regions can be humanized in many different formatsincluding both a full length antibody and a single-chain Fv (scFv).Since the described invention relies on prokaryotic recombinant proteinexpression, a full length antibody structure is not desirable. However,the invention does describe successful humanization of rabbit V-regionsin an antibody format. Regardless of the format, the current inventioninvolves removal of any naturally occurring methionine residues,substituting them with other amino acids. Since methionine residues arefrequently found within framework regions and CDRs of immunoglobulinV-regions, it is necessary to find suitable replacements for theseresidues where they occur without impacting the expression, stability orfunction of the desired protein. This methionine-free scFv can then beoptimized for expression in a methionine auxotrophic bacterial strain,purified, refolded and tested for biologic activity.

Successful humanization and subsequent methionine substitution providespart of a therapeutic vehicle that can be chemically modified byinsertion of a single methionine codon that serves as an insertion sitefor a non-natural amino acid with a chemically reactive site forcovalently linking other complementary molecules such as an activatedPEG moiety. This PEGylated scFv can then be further modified by covalentlinkage to another such scFv through a similarly reactive group at theremaining terminus of the PEG polymer. This bi-specific, PEGylatedproduct can then be purified and refolded to yield a stable,biologically active therapeutic protein.

6.1 Full Length Antibody Humanization Process.

Rabbit-human chimeric monoclonal antibodies can be humanized as fulllength antibodies. This entails the exchange of human VH and VLframework regions for the rabbit frameworks with the retention of therabbit CDRs and often includes retaining particular rabbit frameworkresidues. Just as there are multiple strategies for humanizing rodentV-regions there are other possible methods by which a rabbit-humanchimeric antibody might be partially or fully humanized. Here wedescribe the method used to humanize anti-human IL-6 clone 9C8 in anantibody format. 9C8, a high affinity and high potency chimeric mAb, washumanized by changing the framework regions of the VH and VL to humanframework sequences, with limited back mutation to rabbit frameworksequences.

Humanizing NZW rabbit V-regions was accomplished by first comparingtheir primary amino acid sequence to those found in human V-regions(Altschul et al., 1990). Selection of potentially compatible humanV-region frameworks were made based on sequence similarity withinframework regions (FR1, FR2, FR3 and FR4), sequence length and contentwithin the complementarity determining regions (CDR1, CDR2 and CDR3), aswell as key FR residues that are known to be critical for supporting IgVcanonical loop structures. Using these data human frameworks were chosenfor both light and heavy chain V-regions and the rabbit CDRs weregrafted onto these frameworks as illustrated in FIG. 15 by PCR usingoverlapping oligonucleotide primers (Table 19).

For the humanization of 9C8, the VKappa framework of rabbit was changedto human framework DPK8 VK1, while the VH framework of rabbit changed toDP42 VH3-53 framework of human. All CDR's are rabbit. Two versions (v1and v2) of the heavy chain were made (see below). These two versionsdiffer in the framework region proximal to CDR1 VH (residues H23-30).The endogenous 9C8 rabbit framework region here is amino acid sequenceTVSGIDLS, which was used for v2. For v1, the first two frameworkresidues of this sequence (TV in rabbit) were changed to AA which ishighly conserved in the homologous human VH3 framework positions. Theparental chimeric 9C8 mAb was compared side by side with the humanizedversions, 9C8 mAbv1 and mAbv2 (FIG. 9B). Both versions retained fullactivity.

Framework and CDR1VH Variations in 9C8 v1 & v2: 9C8 v1

FW VH back mutations (H23-30): AASGIDLS (SEQ ID NO. 355)

CDR1 VH (H31-35): SYDMS 9C8 v2

FW-VH back mutations (H23-30): TVSGIDLS (SEQ ID NO. 356)

CDR1 VH: (H31-35) SYDMS

FIG. 9B illustrates a side by side comparison of the parental chimeric9C8 mAb with the humanized 9C8 mAbs (9C8 mAbv1 and 9C8 mAbv2) containingthe 2 different rabbit back mutations proximal to CDR1 of the VH region(at positions H23-H30). The humanized monoclonal antibodies 9C8 mAbv1(containing TV at VH residues 23-24) and 9C8 mAbv2 (AA at 23-24) wereexpressed by transient co-transfection of both heavy and light chainDNAs into HEK293 cells as described previously. The humanized monoclonalantibodies were tested for neutralization of 50 pg/ml IL-6, as indicatedThese changes, TVSGIDLS (mAbv2) or AASGIDLS (mAbv1) do not affectfunctional activity (FIG. 9B). 9C8 mAbv1 was further compared tohumanized mAb 18D4 in FIG. 9C.

TABLE 19Oligonucleotide Primers used to graft rabbit CDRs on selected humanV-region frameworks. AZ_ID SEQ_ID primer sequence scFv-NotF 200GCGATAGCGGCCGCACCACCATGGAGGCTCCC JHXhoR 201GCTATACTCGAGACGGTGACCAGGGTGCCCTGGCCCC DPK8F1 202GACATCCAGTTGACCCAGTCTCCATCCTTTCTGTCTGCATCTGTAGGAGACAG DPK8-AgeF 203GACACAACCGGTGACATCCAGTTGACCCAGTC 9C8-H1F 204GAATCGACCTCAGTAGCTACGACATGAGCTGGGTCCGTCAGGCACCTG 9C8-H1R1 205GTAGCTACTGAGGTCGATTCCAGAAGCTGCACAGGAGAGGCGCAGGG 9C8-H1R2 206GTAGCTACTGAGGTCGATTCCAGAGACAGTACAGGAGAGGCGCAGGG 9C8-H2F 207ACTGATAGTAGCACATACTACGCGAACTGGGCGAAGGGCCGCTTCACCATCAG 9C8-H2R 208GCGTAGTATGTGCTACTATCAGTATAAATGTAGCTCACCCACTCCAGACCC 9C8-H3R1 209GTGTCGAAAGCATAATCGGTACTACCTCTGGCGCAGTAATACACCGCGGTGTC 9C8-H3R2 210GACCAGGGTGCCCTGGCCCCAGAGATCCAACCGAGTGTCGAAAGCATAATCGG 13A8-L2F 211GGGCATCCACTCTGACATCTGGAGTCCCATCAAGGTTC 13A8-L2R 212GACTCCAGATGTCAGAGTGGATGCCCTATAGATCAG 13A8-H2F 213 GCACATGGTACGCGAACTGGG13A8-H2R 214 AGTTCGCGTACCATGTGCTACTATCAG 13A8-EcoF 215GGGACAGAATTCACTCTCACAATCAGC 13A8-EcoR 216 GAGAGTGAATTCTGTCCCAGATCCACTG31A12-L1F 217 GCCAGTGAGGACATCGAGAGCTACCTGGCTTGGTATCAGCAAAAACCAG31A12-L1R 218 AGCTCTCGATGTCCTCACTGGCCTGGCAAGTGATGGTGACTCTGTC 31A12-L2F219 AGTGCATCCACTCTGACCTCTGGCGTCCCATCAAGGTTCAGC 31A12-L2R 220AGAGGTCAGAGTGGATGCACTATAGATCAGGAGCTTAGGG 31A12-L3F 221GTCTCGGTGCTGACGATACCACTACCGTCTTCGGCGGAGGGACCAAGGTG 31A12-L3R 222AGTGGTATCGTCAGCACCGAGACAGTAATAAGTTGCAAAATCTTCAG 31A12-H1F 223GGATTCAGCCTCAGTTCCTATTGGATGACCTGGGTCCGTCAGGCACCTG 31A12-H1R 224ATAGGAACTGAGGCTGAATCCAGAGGCTGTACAGGAGAGGCGCAGGGAC 31A12-H2F 225AGCTCCACATACTATGCATCTTGGGCGAAAGGCCGCTTCACCATCAGCCGC 31A12-H2R 226GATGCATAGTATGTGGAGCTGGTAGCAATAGTGCCCACCCACTCCAAACCC 31A12-H3R1 227AGAGATCTAAGTCATAGTCTGTAGTGAGTCCTCTGGCGCAGTAATACACC 31A12-HXhoR 228GACCGCTCGAGACGGTGACCAGGGTGCCCTGGCCCCAGAGATCTAAGTCATAGTC 45G5-L1F 229AGAGTATTTATAATAACAACCTCTTATCCTGGTATCAGCAAAAACCAGGG 45G5-L1R 230GGATAAGAGGTTGTTATTATAAATACTCTGACTGGACTGGCAAGTGATGGTGAC 45G5-L2F 231GCATCCACTCTGGCATCTGGCGTCCCATCAAGGTTCAGC 45G5-L2R 232GCCAGATGCCAGAGTGGATGCAAAATAGATCAGGAGCTTAGGG 45G5-L3F 233GGCGGTTATGATGATGATGCTGATACTGCTTTCGGCGGAGGGACCAAGGTG 45G5-L3R 234GCATCATCATCATAACCGCCTAGACAGTAATAAGTTGCAAAATCTTCAG 45G5-H1F 235GGATTCTCCCTCAGTGTATATCCAATAAACTGGGTCCGTCAGGCACCTG 45G5-H1R 236GATATACACTGAGGGAGAATCCAGAGACTGTACAGGAGAGGCGCAGGGAC 45G5-H2F 237GTTGATGACACAGCCTACTCAGCCTGGGCGAAAGGCCGCTTCACCATCAGCCGC 45G5-H2R 238GAGTAGGCTGTGTCATCAACATCATTAATAATGCCCACCCACTCCAAACCCTTG 45G5-H3R1 239AGCATCTCCAGCATAAGCATAACTCAAATAACCTCTGGCGCAGTAATACACC 45G5-H3R2 240ACGGTGACCAGGGTGCCCTGGCCCCAGGGATCGAAAGCATCTCCAGCATAAGC 22H8-L1F 241AGACTGTCTATAAGAACAACCTCTTATCCTGGTATCAGCAAAAACCAGGG 22H8-L1R 242GGTTGTTCTTATAGACAGTCTGACTGGACTGGCAAGTGATGGTGACTCTG 22H8-L2F 243GATCTATCTGGCATCCACTCTGGCATCTGGCGTCCCATCAAGGTTCAG 22H8-L2R 244AGATGCCAGAGTGGATGCCAGATAGATCAGGAGCTTAGGG 22H8-L3F 245AGGCGGTTATGATGATGACGCTGATACTGCTTTCGGCGGAGGGACCAAGGTG 22H8-L3R 246GCGTCATCATCATAACCGCCTAGACAGTAATAAGTTGCAAAATCTTCAG 22H8-H1F 247GGATTCTCCCTCAGTACCTATACANTGAACTGGGTCCGTCAGGCACCTG 22H8-H1R 248GTATAGGTACTGAGGGAGAATCCAGAGACTGTACAGGAGAGGCGCAGGGAC 22H8-H2F 249ATGGTGGCACAGCCTACGCGAACTGGGCGAAAGGCCGCTTCACCATCAGCCGC 22H8-H2R 250GCGTAGGCTGTGCCACCATCATAACTAATGGCGCCCACCCACTCCAAACCC 22H8-H3R1 251GAAAGCATCCCCAATATAAGCATAAACATAAAAACCTCTGGCGCAGTAATACACC 22H8-H3R2 252GGTGACCAGGGTGCCCTGGCCCCAGGGATCGAAAGCATCCCCAATATAAGC H82XR 253GCAGGCTGTTGANTTGCAGATACAGGGTGTTC H82XF 254ATCTGCAANTCAACAGCCTGCGTGCCGAGGAC DPK8gsR1 255GCCGCTACCGCCACCACCAGAACCGCCACCGCCTTTGATTTCCACCTTGGTCC DPK8gsR2 256CTGCACCTCGGATCCGCCCCCTCCGGAACCACCGCCGCCGCTACCGCCACCACCAG DP427BamF 257GGGCGGATCCGAGGTGCAGCTGGTGGAG DPK8scfvR 258ACCGCCTTTGATTTCCACCTTGGTCCCTCCGCCGAA

The primers used for amplifying the humanized V-regions code forrestriction enzyme sites identical to those used to capture rabbitV-regions in previous Examples and illustrated in FIG. 10. In thismanner, the humanized light chain was ligated to the Ckappa containingexpression vector and the humanized heavy chain V-region was ligated tothe Cgamma1 containing expression vector and transformed into E. coli asdescribed previously. Isolated colonies were then screened andsequenced.

TABLE 20 Framework Naming SEQ_ID Naming rabbit rabbit VH 9C8 AA 56rabbit v-regions for chimeric ab rabbit rabbit VH 9C8 nuc 57 rabbitv-regions for chimeric ab rabbit rabbit VL 9C8 AA 58 rabbit v-regionsfor chimeric ab rabbit rabbit VH 9C8 nuc 59 rabbit v-regions forchimeric ab rabbit 9C8 VH-CDR1 60 rabbit CDR's rabbit 9C8 VH-CDR2 61rabbit CDR's rabbit 9C8 VH-CDR3 62 rabbit CDR's rabbit 9C8 VK-CDR1 63rabbit CDR's rabbit 9C8 VK-CDR2 64 rabbit CDR's rabbit 9C8 VK-CDR3 65rabbit CDR's VH3-66 humanized_9C8 VH AA 196 v-region for 9C8- v6-LLVH3-67 humanized_9C8 VH nt 197 v-region for 9C8- v6-LL DPK5,6humanized_9C8 VL AA 198 v-region for 9C8- v6-LL DPK5,7 humanized_9C8 VLnt 199 v-region for 9C8- v6-LL rabbit 9C8_Rabbit_scFV 313 fully rabbitscFV rabbit 9C8_Rabbit_scFV 314 fully rabbit scFV VH3- 9C8_humanizedscFV_met-free- 315 9C8v6-LL scFV 66/DKP5,6 nucleotide-mammalian- nucexpression_VH3-66-DK5,6(9C8v6- LL) VH3- 9C8_humanized scFV_met-free- 3169C8v6-LL scFV AA 66/DKP5,6 AA-mammalian-expression_VH3-66-DK5,6(9C8v6-LL) VH3- 9C8_humanized scFV_with- 317 9C8v3-1 scFV AA66/DKP5,6 methionine-AA-mammalian- expression_VH3-66-DK5,6:(9v3-1) VH3-9C8_humanized scFV_with- 318 9C8v3-1 scFV nuc 66/DKP5,6methionine-nuc-mammalian- expression_VH3-66-DK5,6:(9v3-1) VH3-9C8_humanized scFV_with- 319 9C8v3-2 scFV nuc 66/DKP5,6methionine-nucleotide- mammalian-expression_VH3-66- DK5,6:(9v3-2) VH3-9C8_humanized-scFV_with- 320 9C8v3-2 scFV AA 66/DKP5,6methionine-AA-mammalian- expression_VH3-66-DK5,6:(9v3-2) VH3-53/DPK89C8_humanized-VK- 321 9C8-vK-humAb nucleotide_DK8_for-Antibody 1&2 nucVH3-53/DPK8 9C8_humanized-VK- 322 9C8-vK-huMab AA_DK8_for-Antibody 1&2AA VH3-53/DPK8 9C8-Version1_humanized-VH- 323 9C8-vHnucleotide_DP42_for-Antibody humAbv1-nuc VH3-53/DPK89C8-Version1_humanized-VH- 324 9C8-vH humAbv1 AA_DP42_for-Antibody AAVH3-53/DPK8 9C8-Version2_humanized-VH- 325 9C8-vH humAbv2nucleotide_DP42_for-Antibody nuc VH3-53/DPK8 9C8-Version2_humanized-VH-326 9C8-vH humAbv2 AA_DP42_for-Antibody AA

Humanized 9C8 scFvs were derived from the humanized mAbv1 (TV) and mAbv2(AA). The resulting scFvs, retained the rabbit framework 1 residues(23-30) proximal to VH1CDR1 These scFvs also retained the endogenousmethionine residues at H34 and H82. Final versions of the humanized scFvfor 9C8 were then made from these scFvs, but substituting newframeworks, DPK5,6/DP47. These new humanized scFvs, 9C8 Hum scFv v3-1and 9C8 Hum scFv v3-2 showed potent anti IL-6 neutralizing activity(FIG. 9D).

6.2 One Step Humanization of V-Regions and Generation of scFvs fromRabbit-Human Chimeric mAbs

Rabbit V-regions can also be humanized directly in an scFv format.Although the humanization methods used may be generally the same asthose used for humanizing monoclonal antibodies, not all humanizedantibodies are easily converted to an scFv. Moreover, humanization of anantibody carries the requirement to account for Constant regioninteractions with the grafted CDR. The sequences of both heavy and lightchain V-regions were compared to human germline and expressed sequencesusing both V-base (http://vbase.mrc-cpe.cam.ac.uk/) as well as IgBLAST(http://www.ncbi.nlm.nih.gov/) as described in Example 6.1.

The majority of the cloned rabbit VH and VL regions closely matchedmembers of the human IGVH3 (IGHV3-66, IGHV3-49) and IGVK1 (DPK-9)families, respectively although DPK-8 (VK1 Locus L8, V-BASE database)was used as the light chain framework due to the absence of a methionineat position L4 (see section 6.1).

Humanized scFvs were designed to encode a 5′ Not I restriction enzymesite, followed by a Kozak box (Kozak, 1987), an IgVK3 leader (L2), ahuman VK1-JK4 framework, a 20 amino acid flexible (gly4ser)4 linker,human VH3-JH4 framework, and a 3′ Xho I restriction site nested withinthe last two serine residues at the C-terminus of the VH3-FR4 (FIG. 15)All scFv DNAs were constructed by de novo DNA synthesis usingoverlapping DNA oligonucleotide extension (Dillon and Rosen, 1990),digested with Not I and Xho I (NEB, Ipswich, Mass.), isolated on a 1%agarose-TAE gel , excised from the gel and purified using a MinElute GelExtraction kit (Qiagen, wherever, CA) using the manufacturer'sinstructions. This DNA was ligated to Not I-Xho I digested pcDNA 3.1(−)(Invitrogen, Carlsbad, Calif.) using T4 DNA Ligase (NEB, Ipswich,Mass.). The pcDNA3.1(−) vector cassette had been modified to encode ashort proline-rich linker followed by a 6×His tag(gly-pro-pro-pro-pro-his-his-his-his-his-his) in frame with theC-terminus of the scFv. Ligated pcDNA3.1-6_(—)13A8 was transformed intocompetent E. coli TOP 10 (Invitrogen, Carlsbad, Calif.) and selected onLB agar +100 μg/ml carbenicillin plates (Teknova, Hollister, Calif.) at37° C. overnight. From these plates, isolated colonies were picked andinoculated into 2 mls YT broth +100 μg/ml carbenicillin (Teknova) andgrown overnight at 37° C. in a shaking incubator. DNA was isolated fromseveral clones using PureLink Quick Plasmid Miniprep columns(Invitrogen) then screened by restriction digest for the presence of the0.8 Kbp scFv fragment. Clones that gave the correct restriction patternwere then sequenced on an Applied Biosystems 3130 Genetic Analyzer(Applied Biosystems, Foster City, Calif.) after PCR cycle sequencingusing Big Dye Terminator v3.1 kit (ABI) according to manufacturer'sinstructions. The resulting DNA sequences were analyzed and compared totheir reference nucleotide and amino acid sequences using VNTI v 10(Invitrogen).

After sequence confirmation, each scFv was transfected into HEK293 cellsusing Lipofectamine 2000 (Invitrogen) using the manufacturer's protocol.Briefly, the day before transfection, log phase HEK293 cells were platedinto 12 well culture plates (Corning, Lowell, Mass.) at a density of500,000 cells per well in complete media (DMEM+Glutamax+Non-EssentialAmino Acids+Pen-Strep+10% FBS-Life Sciences) and incubated overnight ina 37° C. CO₂ incubator. When the cells were roughly 80% confluent, 4 μgof scFv DNA was diluted in 100 μL Opti-MEM, 4 μL of Lipofectamine 2000was diluted in 100 μL Opti-MEM, then the two dilutions combined into atransfection mix and incubated at room temperature for 20 minutes. Themedia was then removed from the 12 well plates and replaced with 1 mlper well SFM4-Transfectx -293 serum free media (Hyclone, Logan, Utah)and the transfection mix added dropwise to each well . The transfectionplates were returned to the 37° C. CO₂ incubator and grown for 3 daysand tested for functional activity as described in previous Examples.

Anti IL-6 scFvs, humanized using the one step method, retained IL-6neutralization activity when expressed in mammalian cells, as shown for13A8 (FIGS. 11A-B), 28D2 and 9C8 v3-1 (FIG. 11C). Measurement of bindingaffinities by Surface Plasmon resonance (SPR) also demonstratedsuccessful humanization of the 13A8 and 9C8 anti IL-6 scFvs (Table 21).The humanized 13A8 and 9C8 scFvs used for affinity testing contained a6×-Histidine tag and purified from transfected HEK supernatants (usingmethods described in previous examples) and were tested by SPR carriedon essentially as described in Example 2.

TABLE 21 Affinites and potencies of Humanized and Mammalian Expressedanti IL-6 scFvs (prior to methionine substitutions) and their parentalchimeric mAbs. K_(D) EC50 Antibody Ka (M⁻¹s⁻¹) K_(d) (s⁻¹) (pM) (pg/ml)13A8 chimeric mAb 6.33 × 10⁵ 1.38 × 10⁻⁴ 218 34 13A8 humanized 8.76 ×10⁵ 8.64 × 10⁻⁵ 98.2 64 scFv 9C8 chimeric mAb 7.65 × 10⁵ 3.17 × 10⁻⁵ 42400 9C8 humanized scFv 4.47 × 10⁵ 4.03 × 10⁻⁵ 89 319 28D2 chimeric mAb65 28D2 humanized 9.18 × 10⁵ 1.002 × 10⁻⁴  109 43 scFv

Anti IL-23 31A12 humanized scFv, expressed in mammalian cells, retainedIL-23 neutralization activity comparable to the parental mAb towardsboth human and primate IL-23 (FIG. 12). In addition, 31A12 scFv retainspicomolar affinity at least as good as the parental chimeric mAb (Table22).

TABLE 22 Affinity and potency of Humanized and Mammalian Expressed antiIL-23 scFv 31A12, and the parental chimeric mAb. EC50 Antibody K_(a)(M⁻¹s⁻¹) K_(d) (s⁻¹) K_(D) (pM) (pg/ml) 31A12 chimeric mAb 4.79 × 10⁵2.02 × 10⁻⁴ 422 3286 31A12 humanized 7.73 × 10⁵ 7.11 × 10⁻⁵ 92 1368 scFv

45G5 humanized scFv retained potent biological activity against IL-23 asdescribed in FIG. 13.

TABLE 23 Humanized V regions aminoacid and nucleotide sequences: SEQ_IDAnti IL-6 AZ_ID humanized_13A8 VH AA 259 humanized_13A8 VH nt 260humanized_13A8 VL AA 261 humanized_13A8 VL nt 262 humanized_28D2 VH AA263 humanized_28D2 VH nt 264 humanized_28D2 VL AA 265 humanized_28D2 VLnt 266 Anti IL-23 AZ_ID humanized_31A12 VH AA 267 humanized_31A12 VH nt268 humanized_31A12 VL AA 269 humanized_31A12 VL nt 270 Anti IL-12/23AZ_ID humanized_22H8 VH AA 271 humanized_22H8 VH nt 272 humanized_22H8VL AA 273 humanized_22H8 VL nt 274 humanized_45G5 VH AA 275humanized_45G5 VH nt 276 humanized_45G5 VL AA 277 humanized_45G5 VL nt2786.3 Methionine Substitution in Humanized scFv

Human immunoglobulin (Ig) V regions often contain methionine residues inCDR1 of both the light and heavy chain, at relatively conserved residuesin VH-FR3 (human VH3 family, amino acid H82) as well as at position L4of the kappa light chain. Since these humanized scFvs will ultimately belinked covalently using a methionine analog, all methionine residueswithin the mature scFvs must be replaced by another naturally occurringamino acid. This amino acid substitution must have minimal or no impacton either function or stability of the resulting scFv.

To avoid the methionine residue at light chain amino acid position L4,CDRs were grafted into the human germline framework DPK8 (GenBankX93626), which has a leucine residue at that position. To replace theheavy chain methionine residue at position H82, degenerateoligonucleotide primers were designed such that methionine would bechanged to either isoleucine (ile), leucine (leu), valine (val) orphenylalanine (phe). These four amino acids can be found in IgVH regionsfrom other species at position H82 as well as in some expressed humanantibodies. These new methionine-free scFvs were transiently expressedin HEK 293 cells, then neutralization activity compared to those oftheir parental scFv.

Based on potency and expression, methionine-free DNA sequences wereoptimized for expression in E. coli by altering codon usage andpotential secondary structure that could interfere with translationefficiency

To substitute alternate amino acids at VH position H82M, overlappingdegenerate primers (primers 54, 55 above) were designed to introduceleucine, valine, isoleucine or phenylalanine at position H82 by PCRalong with flanking primers (primers 1, 2 above seq ID's 200 and 201respectively). PCR products were cloned as described above and the DNAsequenced to determine which amino acid was encoded by each selectedclone.

The humanized anti IL-6 scFv 13A8 (as several other scFvs) has 2methionine residues at VH positions H34 and H82. These residues werereplaced by PCR using degenerate oligonucleotide primers resulting inthe substitution of methionine with either leucine, isoleucine, valineor phenylalanine as described above. These methionine-free scFvs (Table24 below) were then transfected (Lipofectamine 2000, Invitrogen,Carlsbad, Calif.) into HEK 293 cells using manufacturer's protocol andthe resulting 72 hour supernatants assayed for IL-6 neutralizationcompared to a wild-type parental scFv control supernatant. Nearly allthe replacements resulted in full retention of activity (FIG. 14A-D).

TABLE 24 13A8 scFv methionine substitutions tested clone name H34 H8213A8-MM M M 13A8-FF F F 13A8-FI F I 13A8-FL F L 13A8-FM F M 13A8-FV F V13A8-IF I F 13A8-II I I 13A8-IL I L 13A8-IM I M 13A8-IV I V 13A8-LF L F13A8-LI L I 13A8-LL L L 13A8-LM L M 13A8-LV L V 13A8-VF V F 13A8-VI V I13A8-VL V L 13A8-VM V M 13A8-VV V V

H34L was generally a well tolerated replacement. Replacements of H82with different amino acids, in combination with H34L, in the anti IL-23humanized scFv, 31A12, resulted in full retention of activity, comparedto the parental Met containing scFv (FIG. 14E). In a similar fashion,replacement of 45G5 H82 with L or V, in combination with H34L resultedin full retention of activity compared to the original chimeric mAb(FIG. 14F). Replacement Mets in the 9C8 humanized scFv with H82L andH34L also retained potent activity (FIG. 14G). 22H8 scFv naturally doesnot have a Met residue at position H82. Changing the Met at H34 witheither L or V resulted in full retention of potent IL-23 neutralizingactivity (FIG. 14H). The specific VH mutations for each lead candidateVH region are shown in Table 25×, showing the rabbit back mutations atH23-30, and H49, as well as the H34 and H82 methionine replacements.

For each of the final lead scFv candidates, Leucine was chosen as theamino acid that was substituted at both positions H82 and H34. Leadcandidate scFv DNA sequences were optimized for expression in E. coli bymodifying the codon usage according to those preferred by E. coli. Atthis point, placement of the single Met residue for substitution in vivoby a methionine analog, such as Aha, was investigated. DNA wassynthesized by PCR (as described above) was cloned into appropriateexpression vector such as pQE vectors. The synthetic DNA gene sequencein the expression vectors were confirmed by DNA sequence. Finally, thesynthetic gene in the expression vector was transformed into Methionioneauxotrophic E. coli host such such B834 for recombinant proteinproduction.

TABLE 25X Amino acid mutations in lead humanized rabbit VH sequences VHFramework 1 Back Mutations scFv H23 H24 H25 H26 H27 H28 H29 H30 H34 H49H82 VH3-66 A A S G F T V S M S M 31A12 T A S G F S L S L G L 45G5 T V SG F S L S *na G na 22H8 T V S G F S L S L G L 13A8 T V S G I D L S L G L28D2 T V S G L S L S na G na 9C8 T V S G I D L S L G L These mutationsare either back mutations to the rabbit sequence (H23-30, and H49) orare Met replacements (H34, H82) common to many VH sequences. *na refersto a position that was not a Met in the rabbit or human sequence and didnot require replacement

TABLE 25A ScFv aminoacid sequences scFv AZ_ID SEQ_ID 13A8 scfv Ecoli 28028D2 scfv Ecoli 282 31A2 scfv Ecoli 284 22H8 scfv Ecoli 286 45G5 scfvEcoli 288

TABLE 25B nt sequences adapted to E. Coli expression scFv AZ_ID SEQ_ID13A8 scfv Ecoli 279 28D2 scfv Ecoli 281 31A2 scfv Ecoli 283 22H8 scfvEcoli 285 45G5 scfv Ecoli 287

The scFv must have a single Met residue introduced at the position wherethe non natural amion acid (NNAA) may be incorporated during proteinproduction in E. coli. This NNAA will become the specific site ofbioconjugation. The NNAA of choice for these products isazidohomoalanine (Aha) which allows the use of copper catalyzedcycloaddition bioconjugation. In order to produce the scFv, containingone Aha residue, scFv DNA containing a single methionine codon, wascodon optimized for E. coli expression and synthesized and cloned into amethionine auxotrophic E. coli strain such as B384. Cells were grown tolog phase, transitioned to methionine-free medium containing Aha, andscFv expression was induced by addition of 1 mM IPTG. Inclusion bodies(IB) containing the desired scFv with a functional Aha group at thedesired location were then isolated.

It was determined that the single Methionine residue might be introducedinto the scFv at many possible positions including, but not limited tothe N and C termini, or in the linker connecting the VL and VH domains.All three single Met forms were constructed of the humanized 28D2 andexpressed in E. coli, with an Aha NNAA introduced in place of theMethionine residue, and tested for functional activity. All three formsretained full biological activity (FIG. 15). In addition, they allretained high affinity,

TABLE 26 Affinities of 28D2 with Aha at different positions scFv K_(a)(M⁻¹s⁻¹) K_(d) (s⁻¹) K_(D) (pM) 28D2 9.18 × 10⁵ 1.002 × 10⁻⁴  109 28D2N-Aha 1.46 × 10⁶ 8.99 × 10⁻⁴ 62 28D2-C Aha 1.45 × 10⁶ 7.60 × 10⁻⁵ 5228D2-L Aha 1.30 × 10⁶ 6.88 × 10⁻⁵ 53

Example 7 PEGylation and Refolding of Humanized scFv General Overview ofBispecific Preparations

Bispecific scFvs are constructed by the conjugation of two differentscFv antigen binding domains to each other by way of a linker. Thisstrategy is realized in a two-step process in which each scFv isconjugated to the bifunctional linker. The two scFvs, comprising thebispecific conjugate contain each a single non natural amino acid (Ahaor other) at a position which serves as a specific site of conjugation.The linker can be homo-bifunctional or hetero-bifunctional and contain acomplementary functional group (Alkyne) that is reactive with theunnatural amino acid contained in the scFv (Aha). The reaction schemehas been successfully applied by the inventors to the successfulgeneration of several bispecific scFv, as detailed in the followingexamples (Scheme 1 below).

The linker employed in these examples is PEG (polyethylene glycol). PEGshave several chemical properties which are desirable in a finalbispecific product and solve problems endemic with scFvs. PEGylationimproves protein solubility and increase scFv stability, reducing scFvaggregation and precipitation. In addition, PEGylation has been shown toincrease serum half life of scFv bispecific product. A long and flexiblelinker such as PEG increases the physical separation of the two antibodyfragments, allowing them to refold independently from each other. Thissolves one of the critical problems that occurs in the refolding ofbispecific antigen binding domains linked by genetic fusion, for whichthere often tends to be uncontrolled and undesirable cross linking ofthe two domains.

The use of a PEG linker has additional advantages due to the flexibilityof chemical synthesis. PEG can be easily functionalized to be acomplementary reaction partner with any unnatural amino acid that isincorporated into the scFv proteins. PEG can also be functionalized withmultiple sites of conjugation which enables construction of multivalentprotein hybrids. The PEG functionalization can be made withhomo-bifunctional or hetero-bifunctional PEG's depending on the desiredconjugation chemistry. The structure of PEG can be tailored for linearor branched variations, which can impact pharmacokinetics andbioactivity.

The chemistry used to conjugate scFvs to the linker is orthogonal to the20 natural amino acids. Azide-alkyne copper mediated cycloadditions isused here, in the preparation of scFv-PEG conjugates and bispecifics. Ina typical sequence, an scFv containing azidohomoalanine (Aha) is reactedwith an excess amount of a homo-bifunctional PEG linker functionalizedwith alkynes. The monovalent PEGylated material is purified and then thefree pendant alkyne of the PEG linker undergoes a second copper mediatedazide-alkyne cycloaddition with a second scFv containing Aha to affordthe bispecific.

Overview of Step 1

The first step in the preparation of bispecifics is the site specificPEGylation of an scFv containing a non-natural amino acid, such asazidohomoalanine (Aha), with PEG that is either homo-bifunctional (eg.Bis-alkyne) or hetero-bifunctional (eg at least mono-alkyne). MonovalentPEGylated scFvs are purified by a series of CHT and SEC chromatographyprior to the second step of the process. The monovalent materials arealso assessed for their ability to be refolded. Finally, the refoldedmaterials are evaluated by bioassay for activity.

The PEGylation of scFvs containing the non-natural amino acidazidohomoalanine (Aha) proceeds with an excess of PEG bis-alkyne (2-100equivalents). A variety of PEG molecular weights have been used. Theazide-alkyne cycloaddition used for conjugation is mediated by Copper(I), originating from a copper (I) source such as CuI or derived byreducing a copper (II) source (CuSO4) with a reducing agent such as DTT,cysteine, beta-mercaptoethanol, glutathione, cystamine,tris-carboxyethylphosphine. A ligand such asTris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, TBTA, is alsoincluded in the reaction mixture. Ligands such as TBTA have been shownto stabilize the reactive copper species and improve reaction yields.The reaction pH is held between 3-10 or optionally between 6-9 by theaddition of buffering reagents, such as sodium phosphate buffer, Tris orHEPES. Additional excipients such as SDS may be used to enhance reactionconditions and protein dynamics. The ability to incorporate anon-natural amino acid such as Aha anywhere in the backbone of the scFvsequence, subsequently enables the PEGylation to occur at thispredefined location. In these examples, PEGylation has been demonstratedat the C, and N terminus of the scFv, but could occur at additionalprogrammed locales as well This generalized procedure has beensuccessfully employed to PEGylate several anti IL-6 scFvs and anti IL-23scFvs. (Scheme 2).

Following the reaction, the monovalent PEGylated scFv can be separatedfrom the unreacted scFv and PEG. This prevents the formation of sideproducts in the subsequent bispecific preparation step. To that end, themixture is typically centrifuged or filtered to remove solidparticulates and the solution treated with excess reducing agent such asDTT. The solution then undergoes a series of chromatography steps. Thefirst step in the purification of the monovalent scFv-PEG is loading thereduced reaction mixture onto CHT column which captures the reactingscFv and the scFv-PEG product, but does not bind all unreacted PEG. Thereacting scFv and product scFv-PEG can be partially or fully resolved byphosphate elution from the CHT column. The desired fractions are pooledand subsequently loaded onto a size exclusion column (SEC), which canseparate the residual unreacted scFv . After SEC, the material is ofsufficient purity to be used in the next step or undergo refoldingexperiments.

7.1 Conjugation of anti IL-6 scFvs

7.1.1 28D2c Aha Conjugation to 30 kDa PEG

To a 250 mL glass beaker with magnetic stir bar placed sodium phosphatebuffer (250 mM, pH 7.4, 7.1 mL) and a solution SDS (10% wt/vol, 3.3 mL).A solution of 28D2c Aha (2.81 mg/mL, 1 equiv, 53 mL) and a solution of30K PEG alkyne (NOF, 2 mM, 60 mg/mL, 8.7 equiv, 22 mL) were added. Asolution of TBTA triazole ligand and copper iodide in DMSO (80 mM bothcomponents, 2.8 mL) was added rapidly to effect precipitation. Themixture was allowed to stand for 5 minutes before stirring was started.The mixture was stirred overnight (18 h) and was subsequently assayed bySDS-PAGE (reducing) and laser densitometry, which indicated a yield of56%

The reaction mixture was poured into 50 mL centrifuge tubes andcentrifuged (12,000 g, 10 min). The supernatant was poured onto DTT (1.5g) and stirred under nitrogen for 1 h. Purification was accomplished bya combination of CHT and SEC chromatography.

7.1.2 PEGylation of 28D2c Aha with PEG Bis-Alkyne

To a 50 mL round-bottomed flask with magnetic stirrer was added water(9.7 mL) and a solution of 28D2c Aha (2.58 mg/mL, 1 equiv, 8.7 mL). Tothis solution was added a solution of 20K PEG Bis-alkyne (3 mM, 60mg/mL, 4 equiv, 1 mL). Added TBTA triazole ligand (48 mg) and allowedsolution to stand for a few minutes. A DMSO solution of copper iodide(40 mM, DMSO solution, 1.1 mL) was added, the round-bottomed flask wascapped and the mixture was stirred overnight (16 h). The reactionmixture was analyzed by SDS-PAGE (reducing) and densitometry, whichindicated a yield of 42% .

The reaction mixture was poured into a 50 mL centrifuge tube andcentrifuged (12,000 g, 10 min). The supernatant was added to DTT (462mg) and stirred under nitrogen for 1 h before storage at −20° C.

7.1.3 PEGylation of 13A8n Aha with 20K PEG Bis-Alkyne

In a 400 mL glass beaker with magnetic stir bar was placed sodiumphosphate buffer (50 mM, pH 7.4, 14 mL), a solution of sodiumdodecylsulfate (10% wt/vol, 42 mL) and a solution of dithiothreitol (250mM, 2.7 mL). A solution of 13A8n Aha (3 mg/mL, 1 equiv, 86 mL) and asolution of 20K PEG Bis-Alkyne (3 mM, 60 mg/mL, 26 equiv, 75 mL) wereadded. TBTA triazole ligand (537 mg) was added and the mixture wasallowed to stand without stirring. Copper sulfate solution (80 mM, 6.4mL) was added and the beaker covered with aluminum foil. The mixture wasstirred overnight (16 h) at room temperature. The mixture was evaluatedby SDS-PAGE (reducing) with gel analysis by laser densitometry whichindicated a yield of 69% FIG. 16A .

The reaction mixture was poured into a centrifuge bottle, centrifuged(10000 g, 15 min). Poured off supernatant into 250 mL bottle added DTT(3.4 g) and stirred under nitrogen for 1 h. Further purification wasaccomplished by ceramic hydroxyapatite (CHT-I, Bio-Rad) chromatographyfollowed by size exclusion chromatography (SEC) (Superdex 200).

7.1.4 PEGylation of 13A8c Aha with 20K PEG Bis-Alkyne

In 1000 mL glass bottle with screw cap and magnetic stirrer was placedsodium phosphate buffer (50 mM, pH=7.4, 58 mL), a solution of SDS (10%wt/vol, 112 mL) and a solution of dithiothreitol (250 mM, 7.2 mL). Asolution of the scFv 13A8c Aha (3.5 mg/mL, 1 equiv, 206 mL) and asolution of 20K PEG Bis-Alkyne (3 mM, 60 mg/mL, 25 equiv, 200 mL) wereadded to the bottle. TBTA triazole ligand (1.4 g) was added and themixture was allowed to stand without stirring. After 5 min, a solutionof copper sulfate (80 mM, 17 mL) was added, the bottle was capped andthe mixture stirred a modest speed overnight. The blue-grey solution wasevaluated by SDS-PAGE (reducing) and laser densitometry analysisdetermined the reaction to have a yield of 70% (FIG. 16B)

The reaction mixture was poured into a pair of centrifuge bottles andcentrifuged (10000 g, 15 min). The supernatant was poured off into a 2 Lglass bottle with screw cap. DTT was added (9 g), the vessel wasblanketed with nitrogen and stirred for 1 h. Purification of thereaction mixture was accomplished by a combination of CHT and SECchromatography as in Example 7.1.3.

7.1.5 PEGylation of 13A8c Aha with 40K PEG Bis-Alkyne

In a 500 mL polycarbonate centrifuge bottle with screw cap and magneticstirrer bar was placed a solution of the scFv 13A8cAHA (8.8 mg/mL, 1equiv, 142 mL), a solution of sodium phosphate buffer (500 mM stocksolution, pH=7.4, 23 mL) and a solution of SDS (20% wt/vol stocksolution, 8.8 mL). 40K PEG Bis-Alkyne (9.35 g) was added as a solid tothe stirring solution. The mixture was stirred until all PEG wasdissolved and TBTA triazole ligand (446 mg) was added and the mixturewas allowed to stand without stirring for five minutes. Stirring wasresumed and a fresh solution of cysteine (250 mM stock, 534 uL) wasadded. A solution of copper sulfate (160 mM stock solution, 2.6 mL) wasadded, and the mixture was blanketed with nitrogen and stirred for 4 hwith modest stirring. The reaction mixture was sampled for SDS-PAGE(reducing) with gel analysis by laser densitometry to determine thereaction yield (51%) (FIG. 16C).

The stir bar was removed from the reaction vessel and the mixture wascentrifuged at high speed (10000 g, 15 min). The supernatant was pouredoff into a 500 mL polycarbonate bottle. DTT was added (3 g), the vesselwas blanketed with nitrogen and stirred for 1 h. Purification of thereaction mixture was accomplished by a combination of CHT and SECchromatography as in previous examples.

7.1.6 PEGylation of 13A8L Aha with 20K PEG Bis-Alkyne

In a 250 mL round bottomed flask with magnetic stirrer was placed highlypure water (12.5 mL), a solution of SDS (10% wt/vol stock, 17.3 mL) anda solution of the scFv 13A8L AHA (4.11 mg/mL stock, 26.3 mL). A solutionof 20K PEG Bis-Alkyne (3 mM, 60 mg/mL stock, 30 mL) was added to thereaction mixture. TBTA triazole ligand (214 mg) was added and themixture was allowed to stand without stirring. Stirring was resumed anda solution of dithiothreitol (250 mM stock, 1.08 mL) was added, followedby a solution of copper sulfate (80 mM stock, 2.53 mL). The round bottomwas closed with a septum and stirred overnight. The reaction mixture wasevaluated by SDS-PAGE (reducing) the following day. The resulting gelwas analyzed by laser densitometry analysis, indicating a reaction yieldof 60% (FIG. 16D)

The reaction mixture was transferred to a centrifuge bottle (250 mL) andcentrifuged (12000 g, 15 min). The supernatant was poured off into a 250mL bottle and DTT was added (1.5 g). The vessel was blanketed withnitrogen and stirred until the solids dissolved. Purification of thereaction mixture was accomplished by a combination of CHT and SECchromatography as in previous preparations.

7.2 Reactions with anti-IL-23 scFv Aha7.2.1 IL-23 31A12c Aha scFv Conjugation to 20 kDa PEG-bisalkyne

To a 1000 mL glass bottle with screw cap and magnetic stirrer, wasplaced a solution of sodium phosphate buffer (50 mM, pH=7.4, 65 mL), asolution of SDS (10% solution, 80 mL), and a solution of dithiothreitol(250 mM, 5.1 mL). The solution was stirred gently and a solution ofIL-23-31A12c Aha (pH=7.4, 4 mg/mL, 1 equiv, 121 mL) and a solution of20K PEG Bis-Alkyne (60 mg/mL, 26 equiv, 142 mL) were added. The stirringwas halted and TBTA (1.1 g) was added. The material was allowed tosettle (˜5 min) and a solution of copper sulfate (80 mM, 12 mL) wasadded and stirring resumed. The bottle was capped and the mixturestirred for 16 h at room temperature. The reaction mixture was analyzedby SDS-PAGE (reducing). and the resulting gel was analyzed bydensitometry which indicated a 59% conversion of starting material tothe desired PEGylated product (FIG. 16B).

The reaction mixture was transferred to a centrifuge bottle (500 mL) andcentrifuged (10,000 g, 15 min). The resulting supernatant wastransferred to a sterile polycarbonate bottle, dithiothreitol was added(6.3 g) and the solution stirred for 1 h under nitrogen. Additionalpurification was accomplished by CHT and SEC chromatography as done inexample 7.1.3.

7.2.2 PEGylation of 45G5c Aha with 20K PEG Bis-Alkyne

In a 250 mL round-bottomed flask with magnetic stirrer was placed sodiumphosphate buffer (50 mM, pH 7.4, 74 mL), stirring was started and asolution of dithiothreitol (250 mM, 1.9 mL) was added. A solution of thescFv 45G5c Aha (1.8 mg/mL, 1 equiv, 53 mL) and a solution of 20K PEG bisalkyne (3 mM, 60 mg/mL, 25 equiv, 27 mL) were added to the stirringsolution. Stirring was halted and TBTA triazole ligand (382 mg) wasadded and the mixture allowed to stand for 5 min. Copper sulfatesolution (80 mM, 4.5 mL) was added and the flask was capped with arubber septum. The mixture was stirred on the lowest setting overnight(16 h). The reaction was assayed by SDS-PAGE (reducing gel) and the gelanalyzed by densitometry which indicated a yield of 40% (FIG. 16E)

The reaction mixture was transferred to a centrifuge bottle, centrifugedon tilt rotor (10,000 g, 15 min). The supernatant was poured into a newpolycarbonate bottle, a stir bar and DTT (2.4 g) were added and stirredunder nitrogen for 1 h. Purification was achieved by a CHTchromatography followed size exclusion chromatography as done in example7.1.3.

7.4 Folding:

Folding can occur by taking denatured scFv-PEG (e.g., in 8M urea) andexchanging it (e.g., by dialysis or tangential flow filtration) into apartially denaturing buffer (e.g., 3M urea) that contains a redox system(e.g., cysteine/cystine), followed by exchanging it into non-denaturingbuffer (e.g., phosphate buffered saline).

7.4.1 Folding of 28D2c-PEG

The scFv 28D2 with 30 kDa linear PEG bis alkyne conjugated to the Cterminus was folded. 28D2c-PEG was first purified and buffer exchangedinto a buffer containing 9M urea and dithiothreitol (DTT), pH 7.2.28D2c-PEG was then diluted to starting concentrations of 0.05-1 mg/mLprotein. The starting material was then dialyzed overnight at roomtemperature into a first folding buffer consisting of 3M urea, 30 mMTris pH 8.5, cysteine 2-6 mM, and cystine 1-3 mM. The material was thendialyzed overnight at room temperature into the final buffer consistingof 20 mM sodium phosphate and 150 mM NaCl, pH 7.4.

Refolded material is seen as a monomer both by nonreducing SDS-PAGE andby SEC. The recovery of monomeric 28D2c-PEG was highest at a proteinfolding concentration of 0.05-0.25 mg/mL protein. Similar results wereachieved with cysteine:cystine concentrations ranging from 6:1 to 2:3mM. As a specific example, when the material was folded at 0.1 mg/mLprotein, with 3 mM cystine and 2 mM cysteine, there was 37% monomerrecovery by SEC, the EC50 of the product was 116 pg/mL FIG. 17A, and thebinding affinity measured by SPR (carried on essentially as in example1.4) is given in Table 27.

TABLE 27 Binding affinity of folded 28D2c-PEG refolded by dialysis fromUrea K_(a) (M⁻¹s⁻¹) K_(d) (s⁻¹) K_(D) (pM) 28D2c-PEG 5.07 × 10⁵ 9.78 ×10⁻⁵ 193.3 28D2cAha 1.05 × 10⁶ 7.10 × 10⁻⁵ 67.4

Folding can also occur by taking denatured scFv-PEG and rapidly dilutingit into the partially denaturing buffer and then exchanging it into thenon-denaturing buffer. The starting material for this method cancomprise scFV-PEG denatured in urea or guanidine, or denatured in SDS.28D2c-PEG in a buffer containing 9M urea and DTT, pH 7.2, 1 mg/mL, wasrapidly diluted to 0.05-0.1 mg/mL into a first folding buffer consistingof 3M urea, 30 mM Tris pH 8.5, cysteine 2-6 mM, and cystine 1-3 mM, andthen dialyzed overnight at room temperature in the same buffer. Thematerial was then dialyzed overnight at room temperature into the finalbuffer consisting of 20 mM sodium phosphate and 150 mM NaCl, pH 7.4. Asa specific example, when the material was folded at 0.1 mg/mL, with 2 mMcysteine and 2 mM cystine, there was 38% monomer recovery by SEC, theEC50 of the product was 138 pg/mL.

28D2c-PEG at 0.52 mg/mL protein in buffer containing 0.1% SDS and DTT,pH 7.25 was rapidly diluted into a first folding buffer consisting of 3Murea, 30 mM Tris pH 8.5, cysteine 2-6 mM, and cystine 1-3 mM, and thendialyzed overnight in the same buffer. A 200× dilution was used,reducing the final SDS concentration to 0.0005%. Optionally, the foldingbuffer also contained 400 mM arginine and/or 150 mM NaCl. Alternatively,the folding buffer contained 2 mM glutathinone and 2 mM oxidizedglutathione in lieu of cysteine/cystine. The material was then dialyzedfor 3 days at 5 C into the final buffer consisting of 20 mM sodiumphosphate and 150 mM NaCl, pH 7.4 The material was then concentrated 20fold with a Millipore Centriprep concentrator (10,000 MWCO).

As a specific example, when the material was folded with 3 mM cystineand 2 mM cysteine, there was 20% monomer recovery by SEC, the EC50 ofthe product was 256 pg/mL. IL-6 binding kinetics by these samples wasdetermined by SPR and is given in Table 28. (SPR carried on essentiallyas in Example 1.4)

TABLE 28 Binding affinity of 28D2c-PEG refolded by rapid dilution fromSDS K_(a) (M⁻¹s⁻¹) K_(d) (s⁻¹) K_(D) (pM) 28D2c-PEG 3.26 × 10⁵ 1.04 ×10⁻⁵ 319.4

Folding can also occur by exchange and/or dilution from a startingmaterial denatured in guanidine. 28D2c-PEG was prepared in a buffercontaining 6M guanidine hydrochloride and DTT, pH 8.0. The material wasthen dialyzed into, or rapidly diluted and then dialyzed into, foldingbuffers and then PBS. The protein concentration in the fold was0.05-0.25 mg/mL, and the fold buffer consisted of 3M urea, 30 mM Tris pH8.5, cysteine 2 mM, and cystine 2 mM. Optionally the fold buffer alsocontained 400 mM arginine, and optionally also contained 150 mM NaCl. Asa specific example, when the protein concentration was 0.25 mg/mL, therewas 22% monomer recovery by SEC and the EC50 of the product was 150pg/mL

7.4.2 Folding of other PEGylated scFvs

The scFv's 13A8n, 13A8c, 13A8L and 31A12c, with linear PEG 20 kDaconjugated to the N or C terminus, were also folded by similar methods.The scFv-PEGs were prepared in buffer containing 8M urea and DTT anddiluted to 0.05-0.5 mg/mL total protein. They were then dialyzed at roomtemperature into fold buffer containing 3M urea, 30 mM Tris pH 8.5, 2-6mM cysteine, and 1-3 mM cystine. Alternately, pH 8 or 9, 4M or 2M urea,1% polysorabate 80, and/or dialysis at 4 C were also used. The proteinscould also be folded by dialysis into fold buffer containing no urea,with or without the addition of polysorbate 80. The folding wascompleted by dialysis into PBS, or PBS with the redox system (2-6 mMcysteine and 1-3 mM cystine). Specific examples are given in Table 29.

TABLE 29 Total protein concentration Monomer EC50 in fold CysteineCystine recovery (pg/ Material (mg/mL) (mM) (mM) by SEC mL) 13A8c-PEG200.1 4 2 >95%   93 13A8n-PEG20 0.1 4 2 43% 1976 13A8L-PEG20 0.1 4 2  75%*278 31A12c- 0.1 4 2 55% 1040 PEG20 *13A8L recovery determined by SDSPAGE

Recoveries were best with protein folding at 0.05-0.1 mg/mL. Folding of31A12c-PEG without the presence of urea in the fold buffer resulted inmore disulfide-linked higher molecular weight species in the product.PEGylated scFvs were assessed for stability at different temperaturesand compared to the mammalian expressed unPEGylated scFv. The twoPEGylated species, 13A8c-PEG and 31A12c-PEG, retained all their activityover a 13-20 day period (FIGS. 17B and 17 C).

Tm measurements of the PEGylated scFv's further confirmed the stabilityof these molecules. 31A12-PEG was found to have a Tm of 69.9° C.13A8-PEG was found to have a Tm of 66.1° C.

7.4.3 Folding of unPEGylated scFv's

Similar folding methods could be used for folding unPEGylated scFvs[e.g. 13A8c and 22H8c]. These methods could be useful for folding theproteins prior to conjugation, if desired. As specific examples, 3batches of 13A8c were folded in a buffer containing 3M urea, 4 mMcysteine, 2 mM cystine, 30 mM Tris, pH 8.5, with 0.1 mg/mL totalprotein, followed by dialysis into PBS. The refolding protocol isreproducible, and the monomeric 13A8c recovery yields from 3 batches bySDS-PAGE were 37%, 35%, and 44%, respectively. Refolded unPEGylated22H8c scFv retained high potency compared to the parental mAb.

Example 8 Generation of scFv-PEG-scFv Bispecific Anti IL-6/IL23Conjugates

The next step in the generation of Anti IL-6/Anti IL-23 scFv PEGconjugates is the conjugation of an scFv containing an unnatural aminoacid such as Aha to the scFv-PEG alkyne conjugate prepared in example 7.Following the conjugation reaction, the mixture is purified by acombination of chromatographies prior to undergoing a refolding processto afford the desired scFv-PEG-scFv bispecific. The final materials areassessed for bioactivity and pharmacokinetic properties as well asefficacy in disease models.

The second chemical step in the bispecific preparation is theconjugation of the purified monovalent (scFv-PEG) to the second scFv.The coupling is achieved by the reaction of the free pendant alkyne ofthe monovalent scFv-PEG to Aha of the second scFv via a copper mediatedHuisgen cycloaddition. Several monovalent scFv-PEG conjugates have beenmade successfully and either anti-IL-6-scFv-PEG or anti-IL-23 scFv-PEGcan be used. Likewise, the Aha containing protein can either be ananti-IL-6 scFv or an anti-IL-23 scFv.

For the second reaction, the reaction conditions differ from the coppermediated cycloaddition in the first step. In step one, the reactionconditions employed an excess of PEG-bis alkyne and additives such asSDS to assist the reaction. However, using an excess of alkyne is noteconomically viable or desired from a purification perspective.Therefore, the second step uses a much tighter ratio of alkyne to azide(1:1 to 1:3 alkyne:azide) reaction components. In addition, it was foundthat the second step conjugation works best at higher dilution.Moreover, the TBTA triazole ligand utilized in the first step of theprocess was eventually dropped.

Purification of the reaction mixture proceeds via a mixture ofchromatography, similar to that used in example 7. The Reaction mixtureis first loaded onto a CHT column and eluted with a phosphate gradient.The desired fractions are pooled and then loaded onto a SEC column. Thismaterial can then be further processed for refolding conditions.

In the process described herein, the conjugation precedes the folding.The presence of the PEG linker facilitates the subsequent refolding stepand the scFvs refold independently with minimal interchain crosslinking.interchain crosslinking is a serious impediment often occurring withbispecific constructs linked by genetic fusion and have no PEG linker toprevent the interaction of the antigen binding domains.

8.1 Preparation of Anti IL-23, Anti IL-6 Bispecific 31A12c-PEG-28D2c

In a 1 L glass beaker with magnetic stir bar was placed sodium phosphatebuffer (125 mM, pH 7.4, 486 mL). A solution of 28D2c Aha (4.2 mg/mL, 5.1mL) and a solution of 31A12c-PEG (0.49 mg/mL, 44 mL) were added. Asolution of TBTA triazole ligand and copper iodide (80 mM bothcomponents, 16 mL) was added forming a precipitate. The mixture wasstirred overnight (16 h). The reaction mixture was analyzed by SDS-PAGE(reducing) and densitometry (yield=29%).

The reaction mixture was split into two centrifuge bottles (500 mL) andcentrifuged (10000 g, 30 min) and the supernatant was disposed. To onebottle was added a solution of SDS (8% wt/vol) and a solution of TPPTS(500 mM TPPTS in 1M HEPES, pH 7.4, 25 mL) and sodium phosphate buffer(10 mM, 25 mL). The bottle was nutated and swirled till materials weredissolved or thoroughly suspended. Contents were transferred to thesecond centrifuge bottle/pellet and rinsed out the first centrifugebottle with 2 portions of sodium phosphate buffer (10 mM, 12.5 mL). Thesecond centrifuge bottle was swirled until the pellet was dissolved. Thematerial was centrifuged (10,000 g, 5 min). The supernatant was retainedfor further purification.

8.2 Preparation of Anti IL-23, Anti IL-6 Bispecific 31A12c-PEG-13A8c

To a 2000 mL glass bottle with screw cap and small stir bar was addedwater (814 mL), and a solution of dithiothreitol (250 mM, 12 mL) withgentle stirring. A solution of the scFv 13A8c Aha (0.85 mg/mL, 35 mL)was added followed by a solution of 31A12c-PEG conjugate (0.55 mg/mL, 55mL). A solution of MES buffer (80 mM, pH 7.5, 56 mL) and copper sulfate(80 mM, 28 mL) were added. The bottle was capped and the mixture stirredat the slowest stir speed overnight (16 h). The reaction was analyzed bySDS PAGE (reducing) and densitometry, which indicated a yield of 51%.Two additional 1000 mL reactions were run concurrently with similaryields.

A portion of the pooled reaction mixture (3000 mL) was poured into acentrifuge bottle (˜200 mL per 250 mL bottle) and centrifuged in aspinning bucket centrifuge (Sorvall RC-3BP, 5000 g, 15 min). Thesupernatant was disposed. Additional pooled reaction mixture was addedto the pellet and centrifuged again. The sequence was repeated until allthe reaction mixture had been processed. To the pellet was added 600 mLof the following buffer, 10 mM Phosphates pH=7.4, 2% SDS, and 250 mMDTT. A stir bar was added and the mixture stirred for 30 min, followedby warming to 50 C for 5 min, and then additional stirring at room temp.Solids were disrupted with a glass rod. TPPTS (Strem, 350 mM, pH 7.6, 25mL) was added and the mixture stirred for 1 h at which point all solidsdissolved. The material was passed along for further purification. Acombination of ceramic hydroxyapatite (CHT-I, BioRad) and size exclusion(Superdex 6 prep) chromatography was used to purify the bispecificproduct.

8.3 Preparation of Anti IL-23, anti IL-6 bispecific 13A8c-PEG-31A12c

In a 2000 mL glass bottle with screw cap equipped with a magneticstirrer was placed water (830 mL) and a solution of dithiothreitol (250mM, 12 mL) was added while the solution is gently stirred. A solution ofthe scFv 31A12cAha (0.88 mg/mL, 45 mL) and a solution of the conjugate13A8c-PEG (0.7 mg/mL, 30 mL) were added. MES buffer (80 mM, 56 mL) and asolution of copper sulfate (80 mM, 28.1 mL) were added and the bottlewas capped. Gentle stirring is continued overnight. SDS PAGE analysisand densitometry of the reaction mixture indicated a yield of 48%. Thereaction was run concurrently with two additional 1 L reactions andseven additional 500 mL reactions with an average yield of 49% (FIG.18A).

The pooled 6500 mL reaction volume was processed as follows. Into twocentrifuge bottles (500 mL) placed approximately 450 mL of reactionmixture into each bottle. Centrifuged in swinging bucket centrifuge(5000 g, 15 min). Disposed of supernatant, added additional reactionmixture to each collection centrifuge bottle and centrifuge materialagain. Repeated sequence until all pooled reaction volume had beencentrifuged and the pellet (×2) retained. To each bottle added a stirbar and the following buffer (700 ml): 250 mM DTT, 2% SDS. 10 mM sodiumphosphate buffer. Stirred at room temperature for 30 min. Placed inwater bath (40° C.) and stirred for 10 min. Stirred an additional 30 minat which point no solids remained. The two solutions were pooled beforebeing loaded onto a CHT column. Elution with a phosphate gradient. Thedesired fractions are pooled with additional purification by sizeexclusion column.

8.4 Preparation of Anti IL-23, Anti IL-6 Bispecific 13A8n-PEG-31A12c

In a 2000 mL glass bottle with screw cap equipped with small magneticstirrer was placed water (640 mL) and a solution of DTT (250 mM, 9.6mL). To this mixture was added a solution of 31A12cAha (0.88 mg/mL, 27mL) and a solution of 13A8c-PEG conjugate (0.42 mg/mL, 56 mL). MESbuffer (80 mM, 45 mL) and copper sulfate solution (80 mM, 23 mL) wereadded and the bottle was capped. The mixture was gently stirred at thelowest stirring speed overnight (16 h). SDS-PAGE and densitometryindicated a yield of 47%. Two additional reactions were identicallyprepared as previously described and afforded yields of 51% and 47%respectively upon gel analysis.

The three reaction volumes were combined and processed as follows. Intotwo centrifuge bottles (250 mL) placed approximately 200 mL of reactionmixture into each bottle. Centrifuged in swing bucket centrifuge (5000g, 15 min). Disposed of supernatant, added additional reaction mixtureto each collection centrifuge bottle and centrifuge material again.Repeated sequence until all reaction mixture from the three reactionshas been centrifuged and the pellet retained. To each bottle added astir bar and the following buffer (220 ml): 250 mM DTT, 2% SDS. 10 mMsodium phosphate buffer. Stirred at room temperature for 30 min. Placedin water bath (40° C.) and stirred for 10 min. Solids remained. Removedfrom water bath, added TPPTS solution (250 mM, pH=7.4, 5 mL). Stirred (1h) at which point no solids remained. The solutions were pooled prior toloading onto a CHT column. Elution of the material with a phosphategradient afforded a semi-purified mixture of bispecific and additionalprotein components. Additional purification by SEC afforded the desiredbispecific product.

8.5 Preparation of Anti IL-12/23, Anti IL-6 bispecific 13A8n-PEG-45G5c

In a 2000 mL glass beaker with large magnetic stir bar, was placed water(898 mL) and a solution of DTT (250 mM, 12 mL). A solution of 45G5cAha(0.9 mg/mL, 33 mL) and a solution of 13A8n-PEG (0.98 mg/mL, 30 mL) wereadded. A copper sulfate solution (80 mM, 28 mL) was added and themixture was stirred overnight (16 h). A second identical reaction wasrun in parallel with the previous described reaction. The reactionmixture was assessed by SDS-PAGE (reducing) and densitometry (24%yield—reaction 1 and 25% reaction 2) (FIG. 18B).

Poured approximately 400 mL of reaction mixture in centrifuge bottle(500 mL×2, both reaction mixtures kept separate). Placed in swingingbucket centrifuge, centrifuged (5000 g, 15 min). Disposed ofsupernatant. Repeated sequence until all reaction mixture was processedand only pellet remains. To the pellet added the following buffer (200mL): 20 mM sodium phosphate buffers, 2% SDS, 250 mM DTT. Stirred gentlyfor 30 min. Warmed in water bath (40 C) for 10 min with stirring.Returned to room temperature and stirred till solids dissolved. Thereduced materials were pooled with further purification accomplished byCHT and SEC chromatography.

8.6 Preparation of Anti IL-12/23, Anti IL-6 Bispecific 13A8c-PEG-22H8

In a 2000 mL bottle with screw cap and magnetic stirrer was placed water(950 mL) and a solution of DTT (250 mM, 14 mL) with gentle stirring. Tothis stirred solution was added a solution of the scFv 22H8cAha (0.75mg/mL, 60 mL) and a solution of 13A8c-PEG conjugate (0.69 mg/mL, 35 mL).MES buffer (80 mM, 65 mL) and a solution of copper sulfate (80 mM, 32mL) were added, the bottle was capped and the mixture stirred overnight.SDS PAGE analysis and densitometry indicated a yield of 60% (FIG. 18C).An additional five 1150 mL reactions of same proportions were runconcurrently, with an average yield of 54%.

The combined 6900 mL reaction volume was processed analogously to thatpreviously described. Into two 500 mL centrifuge bottles (500 mL) wasplaced approximately 450 mL (×2) of reaction volume. The mixture wascentrifuged in a swinging bucket centrifuge (5000 g, 15 min). Thesupernatant was disposed, and additional reaction mixture was added toeach collection centrifuge bottle and the centrifuged again. The wasrepeated the entire 6900 mL was processed. To each pellet was added thefollowing buffer (700 mL): 250 mM DTT, 2% SDS. 10 mM sodium phosphatebuffer. Stirred at room temperature for 30 min. The solids were brokenup with a spatula and stirring was resumed for an additional 1 h. Thetwo solutions were combined and loaded onto a CHT column with elution bya phosphate gradient. Additional purification of the semi-purebispecific was accomplished by SEC chromatography.

8.7 Preparation of Anti IL-23, Anti IL-6 Bispecific 13A8c-40 KPEG-31A12c

In a 5000 mL glass bottle with screw cap equipped with a magneticstirrer was placed a sodium phosphate buffer (5 mM stock solution, 2100mL). A solution of the monovalent intermediate 13A8c-40 KPEG (0.34 mg/mLstock, 138 mL) and a solution of the scFv 31A12cAHA (3.2 mg/mL stock,26.1 mL) were added. MES buffer (80 mM stock, 141 mL) and a solution ofdithiothreitol (250 mM stock, 12 mL) were added while the solution wasgently stirred. A solution of copper sulfate (80 mM stock, 70 mL) wasadded and the bottle was capped with gentle stirring continuedovernight. SDS PAGE analysis and densitometry of the reaction mixtureindicated a yield of 58%. The reaction was run concurrently with twoadditional 2.5 L reactions and one additional 1.0 L reactions with anaverage yield of 58% (FIG. 18D).

The pooled 8500 mL reaction volume was centrifuged to collect allsolids. The solids were dissolved in the following workup buffer (1700mL): 250 mM DTT, 2% SDS. 10 mM sodium phosphate buffer. The finalsolution was purified by a combination of CHT and SEC chromatography.

8.8 Preparation of Anti IL-23, Anti IL-6 Bispecific 31A12c-20 KPEG-13A8L

In an 8×30 mM vial with magnetic stirrer was placed water (87 uL) andMES buffer (80 mM stock, 5.6 uL). To this was added a solution of31A12c-20 KPEG (0.550 mg/mL stock, 3.8 uL) and a solution of the scFv13A8LAHA (4.11 mg/mL stock, 0.95 uL). A solution of DTT (250 mM stock,1.2 uL) and a solution of copper sulphate (80 mM stock, 2.8 uL) wereadded, the vial was capped and allowed to stir overnight at roomtemperature. The reaction mixture was sampled for SDS-PAGE the followingday. The resulting gel analyzed by Laser densitometry, indicated a yieldof the bispecific of 37% (FIG. 18E).

8.9 Folding of Bispecific scFvs:

31A12 conjugated to 13A8 via a linear 20 kDa PEG linker (both conjugatedat the C termini) was folded by methods similar to those given above.The bispecific molecule was prepared in a buffer containing 8M urea andDTT, pH 7.3, at 0.05-0.1 mg/mL total protein. The material, at thisstage, might contain some amount of residual unreacted 31A12-PEG scFv inaddition to the bispecific molecule. The material was then folded bydialyzing overnight at room temperature into 3M urea, 30 mM Tris pH 8.5,4 mM cysteine, 2 mM cystine. Optionally, 1% polysorbate 80, 500 mM Tris,or 500 mM Arginine were also added to the fold buffer, or the foldingcould be run at 4 C. The folding reaction was further dialyzed into 20mM sodium phosphate, 150 mM NaCl, pH 7.4 (PBS). As a specific example, 4batches of 0.1 mg/mL of 31A12c-PEG-13A8c were refolded in 3M urea, 30 mMTris pH 8.5, 4 mM cysteine, 2 mM cystine at room temperature, followedby dialysis into PBS. The refolding protocol is reproducible, resultingin similar monomeric bispecific scFv recovery yields and EC50s (Table30, FIGS. 19A and 19B). Monomeric protein was recovered and theresultant molecule retained high bioactivity compared to the parentmolecule. Importantly, the folding worked even in the presence of highamounts of 31A12c-PEG scFv. Moreover, surface plasmon resonance datafurther confirmed the bioactivity of both ends of the bispecific towardsboth IL-6 (13A8) and IL-23 (31A12) targets (Table 31).

TABLE 30 Monomeric bispecific scFv recovery and EC50 from differentbatches Monomer recovery Anti IL-6 EC50 Anti IL-23 EC50 Material by SEC(pg/mL) (pg/mL) 31A12c-PEG-13A8c A 48% 82 2941 31A12c-PEG-13A8c B 23% 641194 31A12c-PEG-13A8c C 41% 136 2038 31A12c-PEG-13A8c D 46% ND ND

TABLE 31 Binding Affinities of the 31A12c-PEG-13A8c Bispecific K_(a)(M⁻¹s⁻¹) K_(d) (s⁻¹) K_(D) (pM) Affinity for IL-6  2.9 × 10⁵ 5.14 × 10⁻⁵214.9 Affinity for IL-23 7.73 × 10⁵ 7.11 × 10⁻⁵ 91.8

The in vivo pharmacokinetics of the bispecific, made with a 20 kDalinear PEG linker was compared to the PK of a naked scFv. The scFv alonewas excreted very rapidly, with a terminal t_(1/2) of about 2 h, Cmaxof500 pg/ml and tmax of 1-2 h, being nearly completely cleared by 8 h(FIG. 20A), The bispecific shows a much longer half life in vivo with aterminal t_(1/2) of about 24 h, Cmaxof 1500 pg/ml, tmax of 24 h, anddetectable levels in the serum at 100 h (FIG. 20B). This improvement inthe pharmacokinetic behavior of the bispecific scFv will make it a muchmore potent and effective therapeutic than a simple scFv.

The same folding methods could be used for PEGylated bispecific13A8n-PEG20-31A12c (PEGylation at the N terminus of 13A8 rather than theC terminus). Two batches of 0.1 mg/mL of protein was folded in 3M urea,30 mM Tris pH 8.5, 4 mM cysteine, 2 mM cystine, followed by dialysisinto PBS. Both batches resulted in >95% monomer recovery by SEC, andEC50s of 1674 and 1691 pg/mL for neutralization of IL-6 by thebispecific compared to an EC50 of 59 pg/ml for the mammalian derived13A8 scFv (FIG. 21), respectively, and 4956 and 3249 pg/mL forneutralization of IL-23, respectively. Monomeric protein was recoveredwith a good yield.

8.8 Refolding of Additional Bispecific scFv Constructs

13A8n-PEG-45G5c was folded by similar methods to those for the 31A12based bispecific. 0.1 mg/mL of protein was folded in 3M urea, 30 mM TrispH 8.5, 4 mM cysteine, 2 mM cystine, followed by dialysis into PBS. Thisresulted in 29% monomer recovery by SEC, and EC50s of 933 pg/mL againstIL-6 and 5,662 pg/mL against IL-23 (FIGS. 22 A and B).

13A8c-PEG-22H8c was folded by similar methods as above. The13A8c-PEG-22H8c was prepared in buffer containing 8M urea and DTT anddiluted to 0.05-0.1 mg/mL total protein. They were then dialyzed at roomtemperature into fold buffer containing 3M urea, 30 mM Tris pH 8.5, 2-6mM cysteine, and 1-3 mM cystine. Alternately, pH 8 or 9, 0.01-1%polysorabte 80, and/or dialysis at 4 C were also used. The folding wascompleted by dialysis into PBS. As a specific example, 0.1 mg/mL of13A8c-PEG-22H8c was folded in 3M urea, 30 mM Tris pH 8.5, 4 mM cysteine,2 mM cystine, 0.05% polysorabte 80, and at room temperature, followed bydialysis into PBS, containing 0.05% polysorbate 80. This resulted in 35%monomer recovery by SEC, and EC50 of 246 pg/mL for neutralization ofIL-6 and 234 pg/mL for neutralization of IL-23 (FIGS. 23 A and B).

13A8c-40 KPEG-31A12c was folded by similar methods to those for the 20Kbispecifics. 0.1 mg/mL of protein was folded in 3M urea, 30 mM Tris pH8.5, 4 mM cysteine, 2 mM cystine at 4° C., followed by dialysis into PBSat 4° C. This resulted in 56.3% recovery by SEC, and EC50s of 137.5pg/mL against IL-6 and 2699 pg/mL against IL-23 (FIGS. 24 A and B). Inaddition, the 13A8c-40 KPEG-31A12c could also be refolded at higherconcentration (0.5 mg/mL) by dialysis into refolding buffer containing0.5M guanidine hydrochloride at both room temperature and 4° C.

The in vivo pharmacokinetics of the 13A8c-40 KPEG-31A12c bispecific, wascompared to the PK of the 13A8c-20 KPEG-31A12c, 13A8c-PEG and 28D2 naked(FIGS. 25A and B). The test articles were dosed subcutaneously in ratsat 1 mg/kg (bispecifics and naked scFv) or 0.5 mg/kg (13A8c-PEG). Bloodwas collected at time intervals after dosing, and serum was assayed forthe presence of test article using the B9 IL-6 neutralization assay. Theresults show that naked scFv is rapidly cleared, while the bispecificsand 13A8c-PEG have a significantly greater half-life and AUC. 13A8c-40KPEG-31A12c also shows a significantly enhanced AUC relative to 13A8c-20KPEG-31A12c (FIG. 25B).

Refolded bispecific conjugates have shown excellent stability for up tosix months at 4° C. The 13A8-PEG-31A12 bispecific has shown consistentpotency in both the anti IL-6 and anti-IL-23 assays. Very littledegradation has been observed in either SDS-PAGE analysis or SECchromatography.

Potency IL6 Potency IL23 Change in purity Time (arbitrary (arbitraryfrom t0 (reducing % monomer (5° C.) units) units) SDS-PAGE) by SEC 0 100100 100% 89% 3 wk 105 93 93% 86% 1 mo 124 123 90% 89% 2 mo 139 86 86%92% 5 mo 84 93 94% 79% 6 mo 84 97 92% 85%

Example 9 Effects of Bispecific scFv on the Generation of Th17 and Th22Cells as Measured In Vitro

Th17 and Th22 T cell subsets can be differentiated in vitro either bystimulation of whole PBMC or purified T cells with anti-CD3 plus antiCD28, or by allogeneic cells in mixed lymphocyte cultures (FIG. 26A).The differentiation of such T cells requires further addition of anumber of key regulatory cytokines that are especially wellcharacterized for Th17 cells. These regulatory cytokines are primarilyderived from myeloid cells and their addition can be replaced with theaddition of myeloid cells along with a compound that stimulates thosecells to release their regulatory cytokines. LPS was used with anti CD3to stimulate whole PBMC to differentiate into Th17 cells. The bestresults were obtained when IL-1 and TGFbeta were also added, as a numberof investigators have previously shown that these cytokines, derivedfrom myeloid cells, promote the differentiation of Th17 from purifiednaïve T cells. Th17 and Th22 T cells can also be differentiated inallogeneic mixed lymphocyte cultures (MLC) with the addition of astimulant to induce the myeloid cells to release their regulatorycytokines. Peptidoglycan was added to the MLC as that stimulant, as itis known to induce the secretion of IL-1, IL-6, TNF and other regulatoryand proinflammatory cytokines. Addition of IL-2 was also required whenthe goal was to study the induction of Th22 cells. PBMC were stimulatedwith anti CD3/28 plus LPS and TGF beta. After 5 days, they wererestimulated with PMA+ ionomycin to induce cytokine secretion andanalyzed by flow cytometry for the expression of IL-17. The percentageof Th17 cells in the PBMC cultures tripled as a result of these cultureconditions (FIG. 26B). Inclusion of IL-6 in combination with IL-23antagonists prevented tripling of Th17 cells. Th22 cells are also seenin the anti CD3/28 stimulation (FIG. 26C). The in vitro stimulation ofhuman T cells in vitro using allogeneic leukocytes also induced highlevels of IL-17 producing T cells (FIG. 26D).

The addition of individual IL-6 and IL-23 antagonists inhibited Th17 andTh22 differentiation in the anti CD3/28 culture system. The combinationof the 2 antagonists, the 31A12 and 13A8 scFvs, was more effective thaneither antagonist alone (FIG. 27). This is also the case for theinhibition of Th17 in MLC by the same antagonists as the previousexperiment (FIG. 28). The 13A8c-20kPEG-31A12c bispecific was more activethan the combination of the parental, chimeric mAbs 13A8 and 31A12, andbetter than either mAb alone, in the inhibition of Th17 cells in MLC(FIG. 29). This demonstrates a beneficial effect obtained through usingthe bivalent bispecific constructs of the present invention.

Example 10 Effects of scFv on the Generation of Th17 and Th22 Cells asMeasured In Vivo

In order to evaluate the inhibition of TH17 and TH22 differentiation invivo, a xenograft model was employed in which human hematopoietic stemcells are transplanted into immunodeficient mice which in turn acquire ahuman immune system. These humanized NOD-scid IL2^(null) (NSG) mice aretransplanted with human skin allogeneic with the human immune cellspopulating the mice (FIG. 30). They are then treated with a mixture ofPEGylated scFv antagonists for IL-6 and IL-23 (13A8c-PEG and31A12c-PEG). The human immune system will then reject this allogeneichuman skin via the differentiation of human T cells into effector cells.The IL-6 and IL-23 antagonists inhibited the differentiation of Th17cells which is one consequence of allogeneic skin transplantation, butthese antagonists did not inhibit the rejection of the skin allograft,reflecting their targeted immunosuppressive effects. Briefly, newbornNSG mice were irradiated and injected with human hematopoietic stemcells derived from umbilical cord blood and then screened forengraftment levels in the peripheral blood at 12 weeks (Brehm et al,2010). Mice that were successfully engrafted were transplanted withhuman allogeneic skin and received 100 μg of anti IL-6 and anti IL-23(13A8c-PEG and 31A12c-PEG) every 2 days. Thirty days after skintransplant, spleens were recovered and single cell suspensions werestimulated with PMA/ionomycin and assayed for intracellular cytokines.CD3+/CD4+ cells were analyzed for IL-17 and IL-22 production by flowcytometry.

In mice that were untreated with cytokine antagonists, very significantlevels of TH17 and TH22 cells developed as shown in the flow cytometryprofiles (FIG. 31A) and in the compiled data representing the numbers ofTh cells in each subset (FIG. 31B). Mice were treated for 30 days, afterskin transplantation, with a combination of anti IL-6 (13A8c scFv-PEG)and anti IL-23 (31A12c-PEG). The differentiation of TH17 and TH22 cellsin treated mice was completely inhibited. These data clearlydemonstrate, for the first time, that IL-6 and IL-23 are required forthe in vivo differentiation of these TH17 and TH22 cells. Furthermore,these data validate this animal model as one which is capable of theelicitation and regulation of human T cell differentiation. Finally,these data demonstrate the effectiveness of the IL-6 and IL-23antagonists used here to completely inhibit the action of thesecytokines in vivo.

Similar results were obtained with the 13A8c-20kPEG-31A12c antiIL-6/anti IL-23 bispecific. As shown in FIG. 32 A-C, the bispecificmolecule is more effective at inhibiting Th17 differentiation than themonovalent anti IL-23 reagent. However, FIG. 32D-L demonstrate that thebispecific is not generally immunosuppressive as leukocyte markers forcell types other than TH17/22 were not significantly reduced.

Example 11 Effects of scFv on the Effector Function of Th17 and Th22Cells as Measured in an Vivo Psoriasis Model

In order to evaluate the inhibition of Th17 effector function in sitesof inflammation, a scid/hu psoriasis model was used, in which humanpsoriatic skin was implanted onto immunodeficient scid mice. The skinengrafts and the psoriatic inflammation persists for up to 2 months. Themice are treated for two weeks with drugs and effects on theinflammation are measured by histological analysis, as shown in FIG. 33,in which the effects of the 13A8c-20kPEG-31A12c anti IL-6/anti IL-23bispecific can be clearly seen in the significant reduction in epidermalthickness. The effect can also be quantitated from the histologicalsections. Comparison of 13A8c-20kPEG-31A12c with its monovalent antiIL-6 inhibitor component, or with the IL-6 antagonist mAb, Tocilizumab(Actemra) demonstrates the significant superiority of13A8c-20kPEG-31A12c over either IL-6 antagonist alone, as determined bysemiquantitiative clinical scoring by a pathologist while the graft isstill on the mouse (FIG. 34 A), or by the quantitative measurement ofepidermal thickness in histological sections as described above (FIG. 34B). In addition, 13A8c-20kPEG-31A12c acts more quickly to inhibitinflammation than Enbrel, a TNF antagonist (FIG. 34 C-D).

Example 12 Effects of Bispecific scFv on the Generation of IL-23Mediated Ear Inflammation Measured In Vivo

When human IL-23 is injected intradermally into the ear of a mouse, theIL-23 will cause inflammation because the human IL-23 can act on themouse IL-23 receptor. The ability of 13A8c-PEG-31A12c to inhibit earinflammation induced by human IL-23 was measured by injecting the eardaily for 4 days with IL-23. Ear swelling was then measured (FIG. 35A).Mice were treated starting a day before and a day after IL-23 treatmentbegan and this treatment effectively blocked the ear swelling (FIG.35B). 13A8c-PEG-31A12c was at least as effective as the IL-12/23antagonist mAb, Stelera (Ustekinumab) as shown in FIG. 35C. Importantly,the treatment of mice with 13A8c-PEG-31A12c , made with either a 20 kDaPEG or a 40 kDa PEG were very effective inhibitors of ear swelling, evenwhen only administered on the day before the IL-23 treatment began (FIG.35D).

Example 13 Epitope Mapping of the IL-23 Specific scFv Component of AZ17

The 31A12 mAbs binds a unique epitope that has not been previouslydescribed. All of the mAbs used bind to human IL-12 (FIG. 36B) eventhough 31A12 and 49B7 are specific for IL-23 inhibition and do notinhibit human IL-12 (FIG. 36B). These data clearly indicate that thesemAbs bind to the p40 chain. 31A12 and 49B7 bind relatively weakly tohuman IL-12 compared to 22H8, which also inhibits IL-12. However, allthree mAbs bind strongly to monkey IL-12 (FIG. 36B) and also inhibitmonkey IL-12 bioactivity (FIG. 36C). Thus 31A12 and 49B7 distinguishhuman and monkey IL-12 activity. It appears that 31A12 and 49B7 see ap40 epitope that is partially masked in human IL-12, and exposed inmonkey IL-12 as well as IL-23 from both species. Moreover, AZ17 does notinhibit the binding of Ustekinumab, a p40 specific mAb that inhibitsboth human IL-12 and IL-23.

REFERENCES

-   Aarden L A, De Groot E R, Schaap O L, Lansdorp P M. Production of    hybridoma growth factor by human monocytes. Eur J Immunol. 1987;    17:1411-6.-   Acosta-Rodriguez E V, Napolitani G, Lanzavecchia A, Sallusto F.    Interleukins 1beta and 6 but not transforming growth factor-beta are    essential for the differentiation of interleukin 17-producing human    T helper cells. Nature Immunol. 2007; 8; 942-9.-   Aggarwal S, Ghilardi N, Xie M H, de Sauvage F J, Gurney A L.    Interleukin-23 promotes a distinct CD4 T cell activation state    characterized by the production of interleukin-17. J Biol Chem.    2003; 278:1910-4.-   Aliahmadi E, Gramlich R, Grützkau A, Hitzler M, Krüger M, Baumgrass    R, Schreiner M, Wittig B, Wanner R, Peiser M. TLR2-activated human    langerhans cells promote Th17 polarization via IL-1beta, TGF-beta    and IL-23. Eur J Immunol. 2009; 39: 1221-30.-   Altschul S F, Gish W, Miller W, Myers E W, Lipman D J. Basic local    alignment search tool. J Mol Biol. 1990; 215: 403-10.-   Brehm M A, Shultz L D, Greiner D L. Humanized mouse models to study    human diseases. Curr Opin Endocrinol Diabetes Obes. 2010 April;    17(2):120-5.-   Dillon P J, Rosen C A. A rapid method for the construction of    synthetic genes using the polymerase chain reaction. Biotechniques.    1990; 9: 298, 300.-   Kabat E A, Wu T T, Perry H M, Gottesman K S, Foeller C. Sequences of    Proteins of Immunological Interest. pp. iii-xxvii, 41-175. National    Institutes of Health, Bethesda, Md., 1992.-   Kozak M. An analysis of 5′-noncoding sequences from 699 vertebrate    messenger RNAs. Nucleic Acids Res. 1987; 15: 8125-48.-   Rader C, Ritter G, Nathan S, Elia M, Gout I, Jungbluth A A, Cohen L    S, Welt S, Old L J, Barbas CF 3rd. The rabbit antibody repertoire as    a novel source for the generation of therapeutic human antibodies. J    Biol Chem. 2000; 275: 13668-76.-   Sehgal D, Johnson G, Wu T T, Mage R G. Generation of the primary    antibody repertoire in rabbits: expression of a diverse set of Igk-V    genes may compensate for limited combinatorial diversity at the    heavy chain locus. Immunogenetics. 1999; 50: 31-42.-   Zubler R H, Erard F, Lees R K, Van Laer M, Mingari C, Moretta L,    MacDonald H R. Mutant EL-4 thymoma cells polyclonally activate    murine and human B cells via direct cell interaction. J Immunol.    1985; 134: 3662-8.

SUPPLEMENTARY REFERENCES

-   Abhinandan K R, Martin A C. Analysis and improvements to Kabat and    structurally correct numbering of antibody variable domains. Mol    Immunol. 2008; 45: 3832-9.-   Allegrucci M, Young-Cooper G O, Alexander C B, Newman B A, Mage R G.    Preferrential rearrangement in normal rabbits of the 3′ VHa allotype    gene that is deleted in Alicia mutants; somatic    hypermutation/conversion may play a major role in generating the    heterogeneity of rabbit heavy chain variable region sequences. Eur J    Immunol. 1991; 21: 411-7.-   Angov E, Hillier C J, Kincaid R L, Lyon J A. Heterologous protein    expression is enhanced by harmonizing the codon usage frequencies of    the target gene with those of the expression host. PLoS One. 2008;    3:e2189.-   Bernstein K E, Lamoyi E, McCartney-Francis N, Mage R G. Sequence of    a cDNA encoding Basilea kappa light chains (K2 isotype) suggests a    possible relationship of protein structure to limited expression. J    Exp Med. 1984; 159: 635-40.-   Better M, Chang C P, Robinson R R, Horwitz A H. Escherichia coli    secretion of an active chimeric antibody fragment. Science. 1988;    240: 1041-3.-   Chothia C, Lesk A M. Canonical structures for the hypervariable    regions of immunoglobulins. J Mol Biol. 1987; 196: 901-17.-   Degryse E. Influence of the second and third codon on the expression    of recombinant hirudin in E. coli. FEBS Lett. 1990; 269: 244-6.-   Déret S, Maissiat C, Aucouturier P, Chomilier J. SUBIM: a program    for analysing the Kabat database and determining the variability    subgroup of a new immunoglobulin sequence. Comput Appl Biosci. 1995;    11: 435-9.-   Eyerich S, Eyerich K, Pennino D, Carbone T, Nasorri F, Pallotta S,    Cianfarani F, Odorisio T, Traidl-Hoffmann C, Behrendt H, Durham S R,    Schmidt-Weber C B, Cavani A. Th22 cells represent a distinct human T    cell subset involved in epidermal immunity and remodeling. J Clin    Invest. 2009; 119:3573-85.-   Gouy M. Codon contexts in enterobacterial and coliphage genes. Mol    Biol Evol. 1987; 4: 426-44.-   Gross G, Mielke C, Hollatz I, Blöcker H, Frank R. RNA primary    sequence or secondary structure in the translational initiation    region controls expression of two variant interferon-beta genes in    Escherichia coli. J Biol Chem. 1990; 265: 17627-36.-   Guisez Y, Robbens J, Remaut E, Fiers W. Folding of the MS2 coat    protein in Escherichia coli is modulated by translational pauses    resulting from mRNA secondary structure and codon usage: a    hypothesis. J Theor Biol. 1993; 162: 243-52.-   Hole N J, Young-Cooper G O, Mage R G. Mapping of the duplicated    rabbit immunoglobulin kappa light chain locus. Eur J Immunol. 1991;    21: 403-9.-   Jones P T, Dear P H, Foote J, Neuberger M S, Winter G. Replacing the    complementarity-determining regions in a human antibody with those    from a mouse. Nature. 1986; 3219: 522-5.-   Kolb H C, Finn M G, Sharpless K B. Click Chemistry: Diverse Chemical    Function from a Few Good Reactions. Angew Chem Int Ed Engl. 2001;    40:2004-2021.-   Khudyakov YuE, Neplyueva V S, Kalinina T I, Smirnov V D. Effect of    structure of the initiator codon on translation in E. coli. FEBS    Lett. 1988; 232: 369-71.-   Knight K L, Becker R S. Molecular basis of the allelic inheritance    of rabbit immunoglobulin VH allotypes: implications for the    generation of antibody diversity. Cell. 1990; 60: 963-70.-   Kreymborg K, Etzensperger R, Dumoutier L, Haak S, Rebollo A, Buch T,    Heppner F L, Renauld J C, Becher B. IL-22 is expressed by Th17 cells    in an IL-23-dependent fashion, but not required for the development    of autoimmune encephalomyelitis. J Immunol. 2007; 12:8098-104.-   Lamoyi E, Mage R G. Lack of K1b9 light chains in Basilea rabbits is    probably due to a mutation in an acceptor site for mRNA splicing. J    Exp Med. 1985; 162: 1149-60.-   Lefranc M P, Giudicelli V, Ginestoux C, Bodmer J, Müller W, Bontrop    R, Lemaitre M, Malik A, Barbie V, Chaume D. IMGT, the international    ImMunoGeneTics database. Nucleic Acids Res. 1999; 27: 209-12.-   Looman A C, Bodlaender J, Comstock L J, Eaton D, Jhurani P, de Boer    H A, van Knippenberg P H. Influence of the codon following the AUG    initiation codon on the expression of a modified lacZ gene in    Escherichia coli. EMBO J. 1987; 6: 2489-92.-   MacCallum R M, Martin A C, Thornton J M. Antibody-antigen    interactions: contact analysis and binding site topography. J Mol    Biol. 1996; 262: 732-45.-   Mage R G. Diversification of rabbit VH genes by gene-conversion-like    and hypermutation mechanisms. Immunol Rev. 1998; 162: 49-54.-   Martin A C, Thornton J M. Structural families in loops of homologous    proteins: automatic classification, modelling and application to    antibodies. J Mol Biol. 1996; 263: 800-15.-   Meldal M, Tornoe C W. Cu-catalyzed Azide-alkyne cycloaddition. Chem.    Rev. 2008; 108:2953-3015.-   Nisonoff A, Rivers M M Recombination of a mixture of univalent    antibody fragments of different specificity Arch Biochem Biophys.    1961; 93:460-2.-   Nograles K E, Zaba L C, Shemer A, Fuentes-Duculan J, Cardinale I,    Kikuchi T, Ramon m, Bergman R, Krueger J G, Guttman-Yassky E. IL-22    producing “T-22” T cells account for the upregaulted IL-22 in atopic    dermatitis despite reduced IL-17-producing Th17 T cells. J. Allergy    Clin. Immunol. 2009; 123: 1244-1252.-   Oresic M, Shalloway D. Specific correlations between relative    synonymous codon usage and protein secondary structure. J Mol Biol.    1998; 281: 31-48.-   Orlandi R, Güssow D H, Jones P T, Winter G. Cloning immunoglobulin    variable domains for expression by the polymerase chain reaction.    Proc Natl Acad Sci USA. 1989; 86: 3833-7.-   Pogulis R J, Vallejo A N, Pease L R. In vitro recombination and    mutagenesis by overlap extension PCR. Methods Mol Biol. 1996; 57:    167-76.-   Sehgal D, Schiaffella E, Anderson A O, Mage R G. Analyses of single    B cells by polymerase chain reaction reveal rearranged VH with    germline sequences in spleens of immunized adult rabbits:    implications for B cell repertoire maintenance and renewal. J    Immunol. 1998; 161: 5347-56.-   Steinberger P, Sutton J K, Rader C, Elia M, Barbas C F 3rd.    Generation and characterization of a recombinant human CCR5-specific    antibody. A phage display approach for rabbit antibody humanization.    J Biol Chem. 2000; 275: 36073-8.-   Sørensen M A, Kurland C G, Pedersen S. Codon usage determines    translation rate in Escherichia coli. J Mol Biol. 1989; 207: 365-77.-   Thanaraj T A, Argos P. Protein secondary structural types are    differentially coded on messenger RNA. Protein Sci. 1996; 5:    1973-83.-   Tiwari A, Sankhyan A, Khanna N, Sinha S. Enhanced periplasmic    expression of high affinity humanized scFv against Hepatitis B    surface antigen by codon optimization. Protein Expr Purif. 2010.    [Epub ahead of print]-   Trinchieri, G, Pflanz S, Kastelein R A. The IL-12 family of    heterodimeric cytokines: New players in the regulation of T cell    responses. Immunity 2003; 19: 641-4.-   Valente C A, Prazeres D M, Cabral J M, Monteiro G A. Translational    features of human alpha 2b interferon production in Escherichia    coli. Appl Environ Microbiol. 2004; 70: 5033-6.-   Wang A, Winblade Nairn N, Johnson R S, Tirrell D A, Grabstein K.    Processing of N-terminal unnatural amino acids in recombinant human    interferon-beta in Escherichia coli. Chembiochem. 2008; 9: 324-30.    PubMed PMID: 18098265.-   Wang Z, Yang D, Wang Q, Li B, Lü Z, Yu J, Zheng H, Fan P, Tang J,    Qian M, et al. High expression of synthetic human interferon-gamma    cDNA in E. coli. Sci China B. 1995; 38: 1084-93.-   Ward E S, Güssow D, Griffiths A D, Jones P T, Winter G. Binding    activities of a repertoire of single immunoglobulin variable domains    secreted from Escherichia coli. Nature. 1989; 341: 544-6.-   Young L, Dong Q. Two-step total gene synthesis method. Nucleic Acids    Res. 2004; 32: e59.-   Zhang W, Xiao W, Wei H, Zhang J, Tian Z. mRNA secondary structure at    start AUG codon is a key limiting factor for human protein    expression in Escherichia coli. Biochem Biophys Res Commun. 2006;    349: 69-78.

1. A bivalent, bispecific construct comprising an anti-IL-6 antibody, orderivative thereof, and an anti-IL-23 antibody, or derivative thereof.2. A bivalent, bispecific construct according to claim 1, wherein theanti-IL-6 antibody, or derivative thereof, is, or is derived from, amonoclonal antibody and/or the anti-IL-23 antibody, or derivativethereof, is, or is derived from, a monoclonal antibody.
 3. A bivalent,bispecific construct according to claim 2, wherein the monoclonalantibodies are human, chimeric or humanized monoclonal antibodies. 4.(canceled)
 5. (canceled)
 6. (canceled)
 7. (canceled)
 8. (canceled)
 9. Abivalent, bispecific construct according to claim 1, wherein theanti-IL-6 antibody, or derivative thereof, comprises a CDR2 regioncomprising the amino acid sequence YIYTDX¹STX²YANWAKG (SEQ ID NO. 335),wherein X¹ is selected from the group consisting of glycine, asparagine,glutamine, cysteine, serine, threonine, and tyrosine; and X² is selectedfrom the group consisting of phenylalanine, tryptophan, and tyrosine;wherein the anti-IL-6, or derivative thereof, comprises a CDR5 regioncomprising the amino acid sequence RX¹STLX²S, wherein X¹ and X² areindependently alanine and threonine.
 10. (canceled)
 11. A bivalent,bispecific construct according to claim 1, wherein the anti-IL-6antibody, or derivative thereof, comprises at least one CDR region whoseamino acid sequence is selected from the group consisting of SEQ ID NOs.10-15.
 12. (canceled)
 13. (canceled)
 14. A bivalent, bispecificconstruct according to claim 1, wherein the anti-IL-6 antibody, orderivative thereof, comprises the amino acid sequences of SEQ ID NO.s10-15.
 15. A bivalent, bispecific construct according to claim 1,wherein the heavy chain of the anti-IL-6 antibody, or derivativethereof, comprises SEQ ID NO. 259 or SEQ ID NO.
 261. 16. (canceled) 17.A bivalent, bispecific construct according to claim 1, wherein theanti-IL-6 antibody, or derivative thereof, is a scFv and wherein theanti-IL-23 antibody, or derivative thereof, is a scFv.
 18. A bivalent,bispecific construct according to claim 1, wherein the anti-IL-6antibody, or derivative thereof, is a scFv comprising (i) a heavy chaincomprising at least one CDR having a sequence selected from the groupconsisting of SEQ ID NOs. 10-12; and (ii) a light chain comprising atleast one CDR having a sequence selected from the group consisting ofSEQ ID NOs. 13-15.
 19. A bivalent, bispecific construct according toclaim 1, wherein the anti-IL-6 antibody, or derivative thereof, is ascFv comprising (i) a heavy chain comprising the amino acid sequence ofSEQ ID NO. 259; and (ii) a light chain comprising the amino acidsequence of SEQ ID NO.
 261. 20. (canceled)
 21. A bivalent, bispecificconstruct according to claim 1, wherein the anti-IL-6 antibody, orderivative thereof, comprises at least one CDR region whose amino acidsequence comprises one or more amino acid additions, deletions orsubstitutions to an amino acid sequence selected from the groupconsisting of SEQ ID NO.s 10-15 or wherein the anti-IL-6 antibody, orderivative thereof, comprises at least one CDR region whose amino acidsequence comprises one or more conservative amino acid substitutions toan amino acid sequence selected from the group consisting of SEQ ID NO.s10-15.
 22. (canceled)
 23. A bivalent, bispecific construct according toclaim 1, wherein the anti-IL-6 antibody, or derivative thereof,comprises at least one CDR region that binds to the same epitope as ananti-IL-6 antibody having CDRs corresponding to the amino acid sequencesof SEQ ID NOs. 10-15.
 24. A bivalent, bispecific construct according toclaim 1, wherein the anti-IL-6 antibody, or derivative thereof, isselected from, or derived from, the group consisting of 13A8, 9H4, 9C8,8C8, 18D4, and 28D2.
 25. A bivalent, bispecific construct according toclaim 1 wherein the anti-IL-23 antibody, or derivative thereof,comprises a CDR2 region comprising the amino acid sequence YYAX¹WAX²G(SEQ ID NO. 337), wherein X¹ is selected from the group consisting ofserine, proline and aspartate, and X² is selected from the groupconsisting of lysine and glutamine or wherein the anti-IL-23 antibody,or derivative thereof, comprises a CDR5 region comprising the amino acidsequence AX¹TLX²S (SEQ ID NO. 338), wherein X¹ is selected from thegroup consisting of serine and alanine X² is selected from the groupconsisting of alanine and threonine.
 26. (canceled)
 27. A bivalent,bispecific construct according to claim 1, wherein the anti-IL-23antibody, or derivative thereof, comprises at least one CDR region whoseamino acid sequence is selected from the group consisting of SEQ ID NOs.90-95.
 28. (canceled)
 29. (canceled)
 30. A bivalent, bispecificconstruct according to claim 1, wherein the anti-IL-6 antibody, orderivative thereof, comprises the amino acid sequences of SEQ ID NOs.90-95.
 31. A bivalent, bispecific construct according to claim 1,wherein the heavy chain of the anti-IL-23 antibody, or derivativethereof, comprises SEQ ID NO. 267 or SEQ ID NO.
 269. 32. (canceled) 33.(canceled)
 34. A bivalent, bispecific construct according to claim 1,wherein the anti-IL-23 antibody, or derivative thereof, is a scFvcomprising (i) a heavy chain comprising at least one CDR having asequence selected from the group consisting of SEQ ID NOs. 90-92; and(ii) a light chain comprising at least one CDR having a sequenceselected from the group consisting of SEQ ID NOs. 93-95.
 35. A bivalent,bispecific construct according to claim 1, wherein the anti-IL-23antibody, or derivative thereof, is a scFv comprising (i) a heavy chaincomprising SEQ ID NO. 267; and (ii) a light chain comprising SEQ ID NO.269.
 36. (canceled)
 37. A bivalent, bispecific construct according toclaim 1, wherein the anti-IL-23 antibody, or derivative thereof,comprises at least one CDR region whose amino acid sequence comprisesone or more amino acid additions, deletions or substitutions to an aminoacid sequence selected from the group consisting of SEQ ID NOs. 90-95 orwherein the anti-IL-23 antibody, or derivative thereof, comprises atleast one CDR region whose amino acid sequence comprises one or moreconservative amino acid substitutions to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs. 90-95.
 38. (canceled)
 39. Abivalent, bispecific construct according to claim 1, wherein theanti-IL-23 antibody, or derivative thereof, comprises at least one CDRregion that binds to the same epitope as an anti-IL-23 antibody havingCDRs corresponding to the amino acid sequences of SEQ ID NOs. 90-95. 40.A bivalent, bispecific construct according to claim 1, wherein theanti-IL-23 antibody, or derivative thereof, is selected from, or derivedfrom, the group consisting of 31A12, 34E11, 35H4, 49B7 and 16C6. 41.(canceled)
 42. (canceled)
 43. (canceled)
 44. (canceled)
 45. (canceled)46. (canceled)
 47. (canceled)
 48. (canceled)
 49. (canceled) 50.(canceled)
 51. (canceled)
 52. (canceled)
 53. (canceled)
 54. (canceled)55. (canceled)
 56. (canceled)
 57. (canceled)
 58. (canceled) 59.(canceled)
 60. (canceled)
 61. (canceled)
 62. A bivalent, bispecificconstruct according to claim 1 wherein both the anti-IL-6 antibody, orderivative thereof, and the anti-IL-23 antibody, or derivative thereof,incorporate one or more non-natural amino acids.
 63. A bivalent,bispecific construct according to claim 62 wherein the anti-IL-6antibody, or derivative thereof, is coupled to the anti-IL-23 antibody,or derivative thereof, through a linker between a non-natural amino acidin each antibody, or derivative thereof.
 64. A bivalent, bispecificconstruct according to claim 63, wherein linker comprises a PEGmolecule.
 65. (canceled)
 66. (canceled)
 67. (canceled)
 68. (canceled)69. (canceled)
 70. (canceled)
 71. (canceled)
 72. (canceled) 73.(canceled)
 74. (canceled)
 75. (canceled)
 76. (canceled)
 77. (canceled)78. (canceled)
 79. (canceled)
 80. (canceled)
 81. (canceled) 82.(canceled)
 83. (canceled)
 84. (canceled)
 85. (canceled)
 86. (canceled)87. (canceled)
 88. (canceled)
 89. A method for producing a bivalent,bispecific construct according to claim 1 comprising: (i) providing ananti-IL-6 antibody, or derivative thereof which is modified byincorporation at least one non-natural amino acid; (ii) providing ananti-IL-23 antibody, or derivative thereof (including ananti-IL-23/IL-12 antibody or derivative thereof), which is modified byincorporation of at least one non-natural amino acid; (iii) reacting themodified anti-IL-6 antibody, or modified derivative thereof, with themodified anti-IL-23 antibody, or modified derivative thereof, such thatthe two are coupled through a linkage between a non-natural amino acidof each portion.
 90. A method according to claim 89, wherein the linkagebetween modified anti-IL-6 antibody, or modified derivative thereof, andthe modified anti-IL-23 antibody, or modified derivative thereof,comprises a linker portion, wherein one end of the linker portion iscoupled to a non-natural amino acid of the modified anti-IL-6 antibody,or modified derivative thereof, and the other end of the linker portionis coupled to a non-natural amino acid of the modified anti-IL-23antibody, or modified derivative thereof.
 91. A method according toclaim 90, wherein the linker portion comprises PEG, a water solublepolymer, polyvinylalcohol, a polysaccharide, a polyalkylene oxide,hydroxyethyl starch, or a polyol.
 92. A method according to claim 89,wherein the non-natural amino acid contains an azide, alkyne, alkene,strained cyclooctyne, strained cycloalkene, cyclopropene, norbornenes oraryl, alkyl or vinyl halide, ketone, aldehyde, cyano, hydrazine, ketals,acetals, hydrazide, alkoxy amine, boronic acid, organotin,organosilicon, beta-silyl alkenyl halide, beta-silyl alkenyl sulfonates,nitrile oxides, pyrones, tetrazine, pyridazine, aryl sulfonates,thiosemicarbazide, semicarbazide, tetrazole, alpha-ketoacid group priorto linkage.
 93. A method according to claim 89, wherein the non-naturalamino acid is azidohomoalanine, homopropargylglycine, homoallylglycine,p-bromophenylalanine, p-iodophenylalanine, azidophenylalanine,acetylphenylalanine or ethynylephenylalanine, amino acids containing aninternal alkene such as trans-crotylalkene, serine allyl ether, allylglycine, propargyl glycine, or vinyl glycine, pyrrolysine,N-sigma-o-azidobenzyloxycarbonyl-L-Lysine (AzZLys),N-sigma-propargyloxycarbonyl-L-Lysine,N-sigma-2-azidoethoxycarbonyl-L-Lysine,N-sigma-tert-butyloxycarbonyl-L-Lysine (BocLys),N-sigma-allyloxycarbonyl-L-Lysine (AlocLys), N-sigma-acetyl-L-Lysine(AcLys), N-sigma-benzyloxycarbonyl-L-Lysine (ZLys),N-sigma-cyclopentyloxycarbonyl-L-Lysine (CycLys),N-sigma-D-prolyl-L-Lysine, N-sigma-nicotinoyl-L-Lysine (NicLys),N-sigma-N-Me-anthraniloyl-L-Lysine (NmaLys), N-sigma-biotinyl-L-Lysine,N-sigma-9-fluorenylmethoxycarbonyl-L-Lysine, N-sigma-methyl-L-Lysine,N-sigma-dimethyl-L-Lysine, N-sigma-trimethyl-L-Lysine,N-sigma-isopropyl-L-Lysine, N-sigma-dansyl-L-Lysine,N-sigma-o,p-dinitrophenyl-L-Lysine, N-sigma-p-toluenesulfonyl-L-Lysine,N-sigma-DL-2-amino-2-carboxyethyl-L-Lysine,N-sigma-phenylpyruvamide-L-Lysine, N-sigma-pyruvamide-L-Lysine. 94.(canceled)
 95. (canceled)
 96. (canceled)
 97. (canceled)
 98. A method oftreating T_(H)17, T_(H)22 or T_(H)1 mediated diseases comprising thestep of administering a therapeutically effective amount of a bivalentbispecific construct according to claim 1 to a patient.
 99. (canceled)100. (canceled)
 101. (canceled)
 102. (canceled)
 103. (canceled) 104.(canceled)
 105. (canceled)
 106. (canceled)
 107. (canceled) 108.(canceled)
 109. (canceled)
 110. (canceled)
 111. (canceled) 112.(canceled)
 113. (canceled)
 114. (canceled)
 115. (canceled) 116.(canceled)
 117. (canceled)
 118. (canceled)
 119. (canceled) 120.(canceled)
 121. (canceled)
 122. (canceled)